Method for enhancing energy production and metabolism in cells

ABSTRACT

The present invention relates to use of a composition comprising D-glyceric acid (DGA), DL-glyceric acid, L-glyceric acid, or hydroxypyruvatic acid and/or their salts or esters. Further, the invention relates to the use of said composition for enhancing direct and indirect mitochondrial metabolism, e.g. the ATP producing electron transport system (ETS), citric acid cycle or tricarboxylic acid cycle, (TCA), and beta oxidation, and also enhancing the shuttling of reducing equivalents from mitochondrial matrix into the cytosol and protein synthesis in the endoplasmic reticulum. Directly related to the above the use of DGA relates also to reducing the formation of reactive oxygen species (ROS). Alleviating, preventing and even healing effects towards extremely wide range of non-communicable diseases materializes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 14/917,764(now U.S. Pat. No. 10,500,176), filed 9 Mar. 2016, which is a U.S.National Stage application of PCT/FI2014/050698 filed 12 Sep. 2014,which claims priority to Finnish patent application 20135927 filed 13Sep. 2013, the entire disclosures of which are hereby incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to use of a composition comprisingD-glyceric acid (DGA), DL-glyceric acid, L-glyceric acid, orhydroxypyruvatic acid and/or their salts or esters. Further, theinvention relates to the use of said composition for enhancing directand indirect mitochondrial metabolism, e.g. the ATP producing electrontransport system (ETS), citric acid cycle or tricarboxylic acid cycle,(TCA), and beta oxidation, and also enhancing the shuttling of reducingequivalents from mitochondrial matrix into the cytosol and proteinsynthesis in the endoplasmic reticulum. Directly related to the abovethe use of DGA relates also to reducing the formation of reactive oxygenspecies (ROS). Alleviating, preventing and even healing effects towardsextremely wide range of non-communicable diseases materializes.Furthermore, the invention relates to a pharmaceutical substance,dietary supplement or nutritive substance comprising said compositions.

BACKGROUND OF THE INVENTION

Non-communicable mitochondrial diseases are becoming an increasingproblem as population gets older and general life expectancy increases.Most often non-communicable mitochondrial diseases arise from somedysfunction of mitochondria itself or dysfunction in communication andcooperation of mitochondria and other cell organelles. Thesedysfunctions can lead to serious pathological conditions such asAlzheimer's disease, Parkinson's disease, cancer, cardiac disease,diabetes, epilepsy, Huntington's disease, and obesity. Mitochondrialmatrix regulates through shuttle mechanisms cytosolic NAD+/NADH-ratio.Additionally in some physiological conditions it can also affectcytosolic NADPH/NADP⁺ ratio. Increased mitochondrial biogenesis has beenproposed as one solution in replacing dysfunctional (damaged) oldmitochondria with new properly functioning mitochondria.

Reactive oxygen species (ROS) or free radicals are producedintracellularly through multiple mechanisms and depending on the celland tissue types, the major sources being NAD(P)H oxidase complexes incell membranes, mitochondria, peroxisomes and endoplasmic reticulum.(NADPH is nicotinamide adenine dinucleotide phosphate in reduced form.)Mitochondria convert energy for the cell into a usable ATP form. Theprocess in which ATP is produced, called oxidative phosphorylation,involves the transport of protons (hydrogen ions) across the innermitochondrial membrane by means of the electron transport chain orbetter described as electron transport system. Various complexes relatedto the ETS are scattered on the inner membrane relatively randomly notas a “chain”. Complexes form a random system guided by greater reductionpotential of next protein complex in the system, i.e. in the ETSelectrons are passed through a series of proteins viaoxidation-reduction reactions with each acceptor protein in the systemhaving greater reduction potential than the previous. The lastdestination for an electron in this system is an oxygen molecule. Smallpart of electrons passing through the ETS escape, and oxygen isprematurely and incompletely reduced to give the superoxide radical.Superoxide is further converted e.g. to H₂O₂. ROS generation is mostwell documented for complex I and complex III.

ROSs are chemically reactive molecules containing oxygen. Examplesinclude oxygen ions, peroxides and nitric oxide (NO). ROS form as anatural byproduct of the normal metabolism of oxygen and have importantroles in cell signaling and homeostasis. However, during times ofenvironmental stress (e.g., UV or heat exposure) or excessive metabolicstress, ROS levels can increase dramatically. This may result insignificant damage to cell structures. Cumulatively, this is known asoxidative stress. ROS are also generated by exogenous sources such asionizing radiation.

Normally, cells defend themselves against ROS damage with enzymes suchas alpha-1-microglobulin, superoxide dismutases, catalases,lactoperoxidases, glutathione peroxidases and peroxiredoxins. Smallmolecule antioxidants such as ascorbic acid (vitamin C), tocopherol(vitamin E), uric acid, and glutathione also play important roles ascellular antioxidants. In a similar manner, polyphenol antioxidantsassist in preventing ROS damage by scavenging free radicals. Antioxidantability of the extracellular spaces, e.g. in plasma, is less efficientthan intracellular ability. According to current knowledge the mostimportant plasma antioxidant in humans is uric acid.

If too much damage is present in mitochondria, a cell undergoesapoptosis or programmed cell death. Bcl-2 proteins are layered on thesurface of the mitochondria, detect damage, and activate a class ofproteins called Bax, which punch holes in the mitochondrial outermembrane, causing cytochrome c to leak out. This cytochrome c binds toApaf-1, or apoptotic protease activating factor-1, which isfree-floating in the cell's cytoplasm. Using ATP as source of energy theApaf-1 and cytochrome c bind together and form apoptosomes. Theapoptosomes bind to and activate caspase-9, another free-floatingprotein. The caspase-9 then cleaves the proteins of the mitochondrialmembrane, causing it to break down and start a chain reaction of proteindenaturation and eventually phagocytosis of the cell.

Metabolic disorders are medical conditions characterized by problemswith an organism's energy metabolism. Excessive nutrition and overweightare frequently related to a metabolic syndrome which has become a majorhealth problem among humans. Metabolic syndrome is a combination of themedical disorders that, when occur together, increase the risk ofdeveloping cardiovascular disease and diabetes. Anabolic and catabolicreactions, regulatory hormones and proteins thereof are in a centralposition in the homeostasis of a human's metabolism. Fat and proteinbiosynthesis are examples of anabolic reactions. Aerobic degradation ofcarbohydrates, fats and carbon skeletons of amino acids represent apathway, wherein oxygen is required in the last resort and whichproduces energy via the respiratory chain of the mitochondria. CoenzymesNAD⁺ (nicotinamide adenine dinucleotide, oxidized) and NADH(nicotinamide adenine dinucleotide, reduced), which regulate the redoxstate of a cell are in a central role in these processes. An excessivereduction of NAD+/NADH results in slow down of TCA, beta oxidation andglycolysis, and it can lead to cellular accumulation of AGEs (advancedglycation end-products). AGEs are proteins or lipids that becomeglycated after exposure to sugars and that cannot be used by normalmetabolic pathways. AGEs are prevalent in the diabetic vasculature andcontribute to the development of atherosclerosis.

In the transition to higher exercise intensity, the rate of adenosinetriphosphate (ATP) hydrolysis is not matched by the transport ofprotons, inorganic phosphate and ADP into the mitochondria.Consequently, there is an increasing dependence on ATP supplied byglycolysis. Under these conditions, there is a greater rate of cytosolicproton release from glycolysis and ATP hydrolysis, the cell bufferingcapacity is eventually exceeded, and acidosis develops (Robergs, 2001).Increased capacity of cytosolic NAD⁺ providing mitochondrial shuttlescan alleviate, postpone, and/or in some cases prevent acidosis.

U.S. Pat. No. 7,666,909 relates to enhancement of alcohol metabolismusing D-glyceric acid. Eriksson et al., 2007 reported thatadministration of ethanol and D-glyceric acid calcium salt to ratsexpedited the metabolism of alcohol. In that scientific paper it washypothesized that the activation of enzymes related to the metabolism ofalcohol and acetaldehyde, i.e. alcohol dehydrogenase and acetaldehydedehydrogenase, and reaction from D-glyceric acid to glycerol andsimultaneous oxidation of 2 NAD⁺ molecules could possibly explain partof the acceleration in ethanol metabolism. Habe et al., 2011 showed inan in vitro study that D-glyceric acid can increase viability ofethanol-dosed gastric cells. Related to that article there seems to bealso a patent that relates to alcohol induced gastrointestinal trackmucous membrane damage and protection against it.

The existing solutions have been found to be ineffective in enhancingaerobic mitochondrial metabolism of carbohydrates, fats and amino acidsas well as treating disorders related to metabolic disorders, especiallyoutside of the gastrointestinal tract. Thus, there still exists a needto provide improved means and methods that are effective in thetreatment and alleviation of metabolic.

SUMMARY OF THE INVENTION

The present invention relates to improved means and methods that areeffective in the treatment and alleviation of metabolic disorders byenhancing mitochondrial aerobic metabolism.

The administration of calcium salt of D-glyceric acid generates aninternal signaling process in cells, organs, and physiological systems,which increases mitochondrial aerobic metabolism and increases theenergy production of cells. In consequence, for example the ability ofmitochondrial shuttle mechanisms (e.g. MA and GP shuttles) to shuttleNAD⁺ from the ETS to cytosol increases. Also beta oxidation of fatsstored as triglycerides increases.

In the present invention the most probable candidate for the locationdependent signaling that increases aerobic energy metabolism is theactivation of GLYCTK1 and/or GLYCTK2 enzymes in the main direction ofDGA and HPA metabolism (see FIG. 1b ). High and prolonged ATP demand,like seen in e.g. endurance exercise, likely eventually also activatesGLYCTK1 and/or GLYCTK2 genes (that can yield ATP). That is likely whyalso DGA and/or HPA administration is able to activate cellularmitochondrial aerobic energy metabolism, including beta oxidation. Allprocesses presented in FIG. 2 are activated as a follow up.

The present inventors have directly by gene expression and mitochondrialbiogenesis analyses shown that said signaling functions in hepatocytes,neurons and peripheral leukocytes. In addition, it has been shown byusing blood tests that e.g. plasma lactate decreases more than 30%,which is a clear and strong indication that the activation of aerobicenergy metabolism occurs also in skeletal muscles, heart and other vitalinner organs. Mitochondrial structures of different cell types differslightly from each other and therefore it is essential to prove directlythat the activation of mitochondria occurs in all cell types. Byactivating Nrf2 pathway, the invention possesses beneficial effects evenin matured red blood cells (RBC) that do not have mitochondria butpossess active Nrf2 pathway.

The gist of the present invention is that mitochondria and mitochondrialenergy metabolism is activated by administrating a compositioncomprising D-glyceric acid, DL-glyceric acid L-glyceric acid orhydroxypyruvatic acid or salt or ester thereof. This leads to activationof PGC-1a/NRF1 and Nrf2/ARE pathways and thus positive effects in theprevention of practically all non-communicative diseases, such ascardiovascular and neurodegenerative diseases, cancer, diabetes,hypertension, auto inflammatory and autoimmune diseases.

The indirect conversion of fats (energy from beta oxidation) to proteinsof muscles tissues (from pyruvate and ammonia from decrease in ureacycle) is a central part of the present invention and this has beenproved in Examples 2.1-2.3, 4 and 5.

An object of the present invention is to provide new means to alleviatethe above mentioned problems.

An object of the invention is to provide a composition comprising one ormore compounds selected from the group consisting of D-glyceric acid,DL-glyceric acid, L-glyceric acid, and hydroxypyruvatic acid and saltsand esters thereof (later referred also as D-glycerate group) for use ina method of enhancing the direct and indirect mitochondrial metabolismand/or excretion of sugars (carbohydrates), fats (lipids) and/or aminoacids. The enhancement is achieved by activating aerobic energymetabolism (the ETS), mitochondrial MA- and GP-shuttles, and byactivating endogenous antioxidant defense mechanism andanti-inflammatory control of the cells (the Nrf2/ARE pathway).Biogenesis of new mitochondria is increased. By enhancing mitochondrialaerobic metabolism in cells and biogenesis of new mitochondria the useof DGA promotes alleviating effect towards non-communicablemitochondrial diseases in cells, tissues/organs and whole physiologicalsystems, e.g. cardiovascular and/or central nervous systems.

An additional object of the use of DGA is to provide substrates forenhancing anaplerotic and anabolic processes like glyceroneogenesis,protein synthesis, and pentose phosphate pathway producingribose-5-phosphate, the precursor of nucleobases adenine and guanine.

An advantage of the innovation is observed in fed and fasting state.Antioxidative state of the cells can be improved directly by: increasingthe amount of reduced ubiquinol (from ubiquinone), and indirectly byincreasing cytosolic NADPH generating capacity internally (PPP) and fromthe mitochondrial matrix. Enhanced energy metabolism and reducedoxidative stress of all cell types can improve whole physiologicalsystems in all organisms. In prior art solutions there is no teachingthat D-glyceric, DL-glyceric acid, L-glyceric acid, and hydroxypyruvaticacid and salts and esters thereof acid can improve antioxidant status ofthe cells.

Another object of the invention is to provide a composition comprisingone or more compounds selected from the group consisting of D-glycericacid, DL-glyceric acid, L-glyceric acid, and hydroxypyruvatic acid andsalts and esters thereof for use in a method of treating or preventing anon-communicable disease or disorder. An object of the invention is alsothe use of said composition for improving general health of subjects inneed.

Another object of the invention is the said composition for use in amethod of reducing weight, in a method of treating or preventing acardiovascular disease, in a method of treating or preventing ametabolic syndrome or a disorder associated with metabolism, in a methodof treating or preventing the aging process of an organism, or in amethod of treating or preventing cancer.

Another object of the present invention is to provide a composition foruse in a method of influencing sugar, fat and/or amino acid metabolismand treating metabolic disorders which comprises a unit dosage formcomprising a therapeutically effective unit dosage of D-glyceric acid,DL-glyceric acid, L-glyceric acid, hydroxypyruvatic acid and salts oresters thereof.

Another object of the invention is to provide a composition comprisingone or more compounds selected from the group consisting of D-glycericacid, DL-glyceric acid, L-glyceric acid, and hydroxypyruvatic acid andsalts and esters thereof for use in a method of enhancing physicaltraining, performance and recovery from exercise.

Another object of the invention is to provide a composition comprisingone or more compounds selected from the group consisting of D-glycericacid, DL-glyceric acid, L-glyceric acid, and hydroxypyruvatic acid andsalts and esters thereof for use as an antioxidant.

Yet another object of the invention is a method of enhancing themetabolism of carbohydrates, and/or fats in a subject comprisingadministering a composition comprising an effective amount of one ormore compounds selected from the group consisting of D-glyceric acid,DL-glyceric acid, L-glyceric acid, and hydroxypyruvatic acid and saltsand esters thereof to a subject in need of preventing dysfunction in thecatabolism of carbohydrates.

An additional object of the invention is an oral, topical, parenteral orinhalable composition for use in a method of prevention of dysfunctionin catabolism of sugars, and enhancing the metabolism fat and/or aminoacid comprising one or more compounds selected from the group consistingof D-glyceric acid, DL-glyceric acid, L-glyceric acid, andhydroxypyruvatic acid and salts and esters thereof. Said composition ise.g. a pharmaceutical preparation.

An additional object of the invention is a method of enhancing thebiosynthesis of phospholipids and medium chain triglycerides enhanced byincreased glyceroneogenesis. Furthermore an additional object of theinnovation is to improve oxygen binding capacity of erythrocytes byimproving their redox-state and enhancing glycolysis. Energy productionand metabolism of erythrocytes increases overall viability and wellbeingof erythrocytes. Antioxidants help also by protecting their membraneintegrity from radical species thus helping to keep their discoidalshape, and to increase oxygen releasing capacity of hemoglobin moleculesby increasing 2,3-bisphosphoglycerate formation by increasingintracellular pH(=a follow up of enhanced conversion of NAHD+H⁺ intoNAD⁺ and possibly of proton exporting capacity).

Innovation enhances wellbeing and viability of all cell types that useglycolysis and/or beta oxidation and citric acid cycle and the ETS intheir metabolism and energy production, e.g. hepatocytes, myocytes,skeletal myotubes, erythrocytes, adipocytes, neurons and glial cells.

Organs and tissue types that benefit from the administration of thecompounds of D-glycerate group are: liver, kidneys, pancreas, spleen,heart and skeletal muscles, cardiovascular system, brains and nervoussystem. An advantage of using the compounds of D-glycerate group comesfrom four main sources: 1) from improving the redox state of all cells,2) increase in metabolic flux/diuretic effects (lower blood sugar, fatsand sodium levels), 3) positive antioxidant effects, and 4) increase inmitochondrial biogenesis and energy metabolism.

An advantage of the present invention is the use of a compositioncomprising D-glyceric acid, DL-glyceric acid, L-glyceric acid, orhydroxypyruvatic acid and/or their salts or esters to enhancecommunication and cooperation of mitochondria and other cell organellesand the use of said composition to increase the biogenesis of newmitochondria. Such use has not been disclosed previously.

Another advantage of the invention is the use of a compositioncomprising D-glyceric acid, DL-glyceric acid, L-glyceric acid, orhydroxypyruvatic acid and/or their salts or esters to enhance endogenouscellular antioxidant defense.

Still another advantage of the invention is the incorporation of alsohydroxypyruvatic acid. Reversible reduction and oxidation reactionsbetween D-glyceric and hydroxypyruvatic are an important part in thelong term efficacy of their use.

The present innovation does not relate to alcohol metabolism and/orgastrointestinal track.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a depicts the structure of mitochondrial electron transport system(ETS) on the inner mitochondrial membrane (IMM) and the location of thecitric acid cycle (TCA) and ATP synthase inside the mitochondrialmatrix. Final catabolism of carbohydrates and fatty acids andcarbohydrate parts of amino acids into carbon dioxide (CO₂) and water(H₂O) occur in the TCA and ETS. FIG. 1 furthermore depictsmalate-aspartate (MA)-shuttle and glycerol phosphate-shuttle(GP-shuttle). These shuttle mechanisms are needed because IMM isimpermeable for NADH and NAD⁺. MA-shuttles transport NAD⁺ from thematrix to the inter membrane space (IMS)/cytosol. Used graph is modifiedfrom a publicly available graphwww.studyblue.com/notes/note/n/biologymetabolism/deck/801583.Abbreviations: MA-shuttle, malate-aspartate shuttle (MAL-ASP-shuttle);GP-shuttle, glycerol-phosphate shuttle; G-3-, glycerol phosphate; DHAP,dihydroxyacetone phosphate; TCA, tricarboxylic acid cycle; ETS, electrontransport system; CI (or I), Complex I of the ETS; CII (or II), ComplexII of the ETS, CIII (or III), Complex III of the ETS; CIV (or IV),Complex IV of the ETS; NAD+, nicotinamide adenine dinucleotide,oxidized; NADH, nicotinamide adenine dinucleotide, reduced; FAD, flavindinucleotide, oxidized; FADH2, flavin dinucleotide, reduced; Q,ubiquinone, oxidized; QH2, ubiquinol (fully) reduced.

FIG. 1b depicts some important metabolic routes for D-glycerate (DGA)and hydroxypyruvate (HPA). Reactions catalyzed by D-glyceratedehydrogenase (DGDH) and hydroxypyruvate reductase (GRHPR) occur incytosol/IMS and likely also in some other favorable spaces in other cellorganelles or compartments but not in the mitochondrial matrix.Cytosolic location applies also to D-glycerate kinases (GLYCTK1,GLYCTK2) that relate DGA directly to major cytosolic pathways, i.e.glycolysis and gluco-/glyceroneogenesis. Abbreviations: GDP2,mitochondrial glycerol-phosphate dehydrogenase; GDP1, cytosolicglycerol-phosphate dehydrogenase; GLYCTK1, Glycerate 1-kinase; GLYCTK2,Glycerate 2-kinase; DGDH, D-glycerate dehydrogenase; GRHPR,glyoxylate/hydroxypyruvate reductase; AGXT1,2, alanine-glyoxylateaminotransferase 1 or alanine-glyoxylate aminotransferase 2; PYR,Pyruvate; OAA, oxaloacetate (OAA); PEP, phosphoenolpyruvate; MAL,malate; SER, L-serine; GLYO, glyoxylate; GLY, glycine; 3P-DGA, glyceratephosphate; 3P-HPA, hydroxypyruvate phosphate; 3P-SER, serine phosphate.

FIG. 2 describes major metabolic pathways that the use of DGA activates.Virtuous cycle of enhanced energy metabolism and endogenous antioxidantdefenses with increased mitochondrial activity and mitochondrialbiogenesis and increased pyruvate concentration with less ER stresscreates wide range of specific and also pleiotropic therapeutic effectsthat can alleviate, prevent or even heal basically all non-communicablediseases related to dysfunction in energy metabolism, increased ROSformation, and/or unregulated anti-inflammatory disorders.Abbreviations: PGC-1α, Peroxisome proliferator-activated receptor gamma(PPAR-γ) coactivator 1-alpha; Nrf2, nuclear factor-2 erythroid relatedfactor-2; AREs, antioxidant response elements; NRF1, nuclear respiratoryfactor 1; HO4, inducible herne oxygenase; SIRT1, sirtuin (silent matingtype information regulation 2 homolog) 1; MT-CO1, mitochondriallyencoded cytochrome c oxidase I; Keap1, Kelch-like ECH-associated protein1; NF-kB, Nuclear factor kappa-light-chain-enhancer of activated Bcells; PERK, RNA-dependent protein kinase (PKR)-like ER kinase.

FIG. 3a is a schematic representation of some important reactions thatthe use of DGA facilitates during fasting. Abbreviations: Gene relatedto complex III (MT-CYB), MT-CYB=Ubiquinol Cytochrome c Reductase; Generelated to complex IV (COX1), COX1=mitochondrially encoded cytochrome coxidase I; ME, malic enzyme; MCTs, monocarboxylate transporters; LAC,lactate; aKG, alfa-ketoglutarate; ASP, aspartate.

FIG. 3b is a schematic representation of some important reactions thatthe use of DGA facilitates during fed state. Abbreviations: CIC,mitochondrial citrate iso-citrate carrier; GLUC, glucose; GLUT4, glucosetransporter 4; IR, insulin resistance; ROS, radical oxygen species;G-6-P, glucose-6-phosphate; GSH, glutathione; SOD2, superoxidedismutase.

FIG. 3c depicts the involvement of mitochondria in cell death caused byNMDA stimulation (excitotoxic insult) in neurons. Source of the graph:M. Flint Beal, Energetics in the pathogenesis of neurodegenerativediseases. Trends in Neuroscience, Volume 23, Issue 7 pp. 279-33, 2000.Abbreviations: NMDA receptor, N-Methyl-D-aspartic acid receptor; Cytc,cytochrome c; NOS, (neuronal) nitric oxide synthase; NO, nitric oxide;ONOO⁻, Peroxynitrite.

FIG. 3d depicts one mechanism of action of the DGA and/or HPA use in redblood cells (RBC or erythrocytes). Abbreviations: 3-P-GA,glyceraldehydephosphate; 1,3-BPG, 1,3-bisphosphoglycerate; 2,3-BPG,2,3-bisphosphoglycerate; BPM, bisphosphoglycerate mutase.

FIG. 4 depicts some of the NADPH dependent pathways that the use of DGAactivates and/or can also down regulate. Most of these NADPH dependentpathways and genes are also directly or indirectly related to cellularantioxidant defenses, i.e. Nrf2/ARE related genes and pathways. Ingeneral Nrf2 regulated genes can be divided into three categories: (1)antioxidants, (2) anti-inflammatory, and (3) genes related tomitochondrial biogenesis/protection. Abbreviations: G6PD,glucose-6-phosphate dehydrogenase; 6PGD, 6-Phosphogluconatedehydrogenase; NO, nitric oxide; iNOS, inducible NOS, eNOS, endotheliaNOS; R-5-P, ribose-5-phosphate; 5-PRA, β-5-phosphorybosylamine; L-Arg,L-arginine; CO, carbon monoxide; Fe, iron; DHA, dehydroascorbate.

FIG. 5 depicts results from human hepatocytes (male donor CDP) cellculture study. It clearly demonstrates that under moderate metabolicstress (1.5 hours after addition of new medium) DGA reduces ROSproduction significantly compared to 0 dose/control in standard highnutrition medium. (Later high nutrition medium from Celsis is called“High Medium”.) Furthermore the reduction in ROS is DGA dose dependent.Significant efficacy is reached already with 1.4 μM concentration ofDGA, and on the other hand 0.14 μM concentration had no effect. BiggestROS scavenging effect is seen in 14 μM (14 μM dose equals 2 μg/mlconcentration). In equimolar comparisons against other known efficientantioxidants (vitamin E, glutathione, and vitamin C) DGA seems to besuperior or at least as good ROS scavenger as this peer group. Molecularweight of all others is greater than DGA and thus also the weight ofequimolar dose. Cell viabilities were rather volatile in different DGAdoses maybe reflecting some cell signaling differences in respect toapoptosis. In general no tendency for big deviations in viabilitycompared to the peer group, thus this analyses gives relatively accuratepicture on ROS scavenging abilities also when analyzing ROS per viablecell. Experimental study setup and some analysis on the statisticalsignificance of the results is described in example 1. In here and inall following graphs sign “*” indicates statistically significantdifference compared to the control (p-value is clearly less than 5%),and “**” statistically very significant difference compared to thecontrol (p-value clearly less than 1%).

FIGS. 6a and 6b depict results from human cell culture studies, whichdemonstrate that under moderate metabolic stress DGA reduces ROSproduction significantly compared to control/0 dose both in standardHigh Medium (FIG. 6a ) as well as in High Medium+0.75 mM palmitic aciddiet (FIG. 6b ) for female donor JGM. (Later high nutrition medium iscalled “High Medium”.) Furthermore DGA seems to be superior antioxidantin equimolar comparison against most other currently known bestantioxidants: vitamin E, glutathione, vitamin C and morin dehydrate.(Molecular weight of all others is greater than DGA and thus also theweight of equimolar dose.) In High Medium only the viabilities of thecell were relatively similar, very small decrease, for all groups exceptfor vitamin E (trolox) and thus the direct comparison of ROS readings iswell justified. Vitamin E decreased viability some 23% and thus the ROSper viable cell was greater for vitamin E than FIG. 6 shows.Experimental setup (for this study 4) and analysis on the statisticalsignificance of the results is described in Example 1.

FIG. 6a shows ROS in JGM (female donor), 20+20+1.5 h, equimolarcomparisons to other best antioxidants in High Medium only. DGA 14 μM=2μg/ml of DGA.

FIG. 6b shows ROS in JGM, 20+20+1.5 h, equimolar comparisons to bestother antioxidants, High Medium and 0.75 mM of Palmitic acid. DGA 14μM=2 μg/ml of DGA.

FIG. 7 depicts that DGA reduces ROS per viable compared to control/0dose in primary human hepatocytes in study 1 for DOD (male donor). ROSper viable cell, DOD (male donor), 24+24+1.5 h, High Medium and HighMedium+Sucrose. Results are measured under moderate metabolic stress. 2DGA=2 μg/ml of DGA=14 μM of DG. 20 DGA=20 μg/ml of DGA=140 μM of DGA.Experimental setup for study 1 and analysis on the statisticalsignificance of the results is described in Example 1

FIG. 8 depicts that DGA reduces ROS per viable cell compared to controlin primary human hepatocytes in study 1 for YJM (female donor). ROS perviable cell, YJM (female donor), 24+24+2 h, High Medium and HighMedium+Sucrose. Results are measured under moderate metabolic stress.Experimental setup for study 1 is described in Example 1.

FIG. 9 depicts that DGA can increases viability of primary humanhepatocytes in study 1 compared to 0 doses for YJM (female donor) andDOD (male donor). Results are measured under moderate metabolic stress.Experimental setup and analysis on the statistical significance of theresults is described in Example 1.

FIG. 9a : VIABILITY DOD, 24+24+1.5 h, High Medium only

FIG. 9b : VIABILITY YJM, 24+24+2 h, High Medium only

FIG. 9c : VIABILITY DOD, 24+24+1.5 h, High Medium+sucrose

FIG. 9d : VIABILITY YJM, 24+24+2 h, High Medium+sucrose

FIG. 10 depicts in study 1 starving diet test, i.e. no addition orchange of medium during 48 hours, that DGA decreases the viability ofhepatocytes compared to control (two upper graphs). Increase ofviability shown in FIG. 9 with optimal nutrition and decrease of theviability of same donors in starving diets indicate clearly that DGAincreases metabolic flux. In study 2 (male donor CDP/lower graph with 3diets)) and normal change of medium, i.e. normal/optimal nutritionalconditions, DGA again increases the viability of hepatocytes in almostall diets compared to 0 dose. It should be noted also that viabilitydecreases with 0.4 DGA dose and Hi Medium+palmitic acid and this declineis statistically significant. Excessive increases of viability (ordecreases) are not normal and they also indicate some kind of excessiveincrease of metabolism in the cells. In vitro cells/tissues can'tcontrol the stimulating effect of DGA, unlike in vivo (FIG. 5 and Table3). Experimental set up for study 2 is described in Example 1.

FIG. 10a : VIABILITY DOD, 48+1.5 h, Starving Diet

FIG. 10b : VIABILITY, YJM, 48 2 h, Starving Diet

FIG. 10c : Viability, CDP (male donor), 20+20+1.5 h, various diets withHigh Medium.

FIG. 11 depicts that DGA decreases ROS compared to 0 dose in study 2(two upper graphs). In study 3 (lower left hand graph) also HPA and LGAdecrease ROS compared to 0 dose. Further in study 3 (lower right handgraph) HPA and LGA increase viability compared to 0 dose. Results aremeasured under moderate metabolic stress. Experimental setup for study 2and 3 is described in Example 1.

FIG. 11 a: ROS, JGM, 20+20+1.5 h, High Medium +0.75 mM Palmitic Acid

FIG. 11 b: ROS, CDP (male), 20+20 +1.5 h, High Medium only

FIG. 11c : ROS, YJM, 20+20+1.5 h, High Medium+0.75 mM Palmitic acid.(LGA 14 =2 μg/ml of LGA)

FIG. 11 d: Viability, YJM, 20+20+1.5 h, High Medium+0.75 mM Palmiticacid. (LGA 14=2 μg/ml of LGA)

FIG. 12 depicts that hypertension was measured in two study subjects outof eight (subject 4 and 5). Subject 5 was the only person with clearlyelevated blood pressure. (For other study subjects 1-3 and 6-8 bloodpressures were at normal level.) Subject 5 blood pressure was firstobserved without any treatment for 6 days (from 20 Jan. to 26 Jan.2013). After that subject 5 received twice a day 4 mg/kg of DGA mixed towater for 10 days. As can be seen in left hand graph during theadministration systolic blood pressure was lowered from roughly 180 tosome 160, and diastolic blood pressure from 102 to some 90. Subject 4received 1×4 mg/k/day of DGA for 3 weeks. Her blood pressure declinedalso somewhat.

FIG. 12a : Hypertension, subject 5 (averages of three measurements. DGAdosing started on 26th of January and ended on 3rd of February. Dose 2×4mg/kg/day).

FIG. 12b : Hypertension, subject 4 (averages of three measurements.Subject 4 received 1×4 mg/kg/day of DGA for 3 weeks).

FIG. 13 “Neuronal viability/baseline LDH release(=LDH(total-leaked))after 96 h treatment with DGAcs in Calorie Restriction (at 7DIV+24 h).”Cell treatment according to the protocol renewed only 25% of the mediumduring SDIV and 6DIV, meaning that neurons received only very smallamounts of new nutrition. De facto the cell culture was under severecalorie restrictions (CR). This is similar to hepatocytes in “StarvingDiet” in Example 1. CR caused dose dependent viability loss also inneurons. Likely explanation is also the same as in hepatocytes: DGAcsadministration increases metabolic (anabolic and catabolic) activity inneurons. Anabolic reactions need a lot of energy thus (aerobic) ATPproduction of neurons and consumption is increased. In a situation withsubnormal source of energy (nutrition) this set up leads to enhancedcell cycle control and programmed cell death (apoptosis).

In FIGS. 13 and 14 a and 14 b error bars are +/−SEM like in FIG. 5, i.e.not stds like in other graphs.

FIGS. 14a and 14b depict the protection by the use of DGA against NMDAstimulated excitotoxic injury in rat cortical neurons. FIG. 14a showsViability at 7 DIV+24 h after 1 h NMDA stimulation at 7DIV. FIG. 14bshows Viability after 24 hours with 1 h NMDA stimulation, indexed to 0NMDA.

DGAcs treatment induces very clear and significant protection againstNMDA-induced excitotoxity in both 25 μM NMDA and 50 μM NMDA group whencell death before and at the start of the test is taken into account(FIG. 14b ). Even without correction for CR induced viability lossbefore the NMDA treatment, DGAcs administration induces very clearprotection against NMDA-induced excitotoxity in 50 μM NMDA group (FIG.14.a). In FIG. 14a “Baseline/NMDA 0” group is the same as in FIG. 13Sign “*” indicates statistically significant difference compared to thecontrol (p-value is clearly less than 5%), and “**” statistically verysignificant difference compared to the control (p-value clearly lessthan 1%).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on studies related to enhancing theenergy production and the metabolism of fats (lipids) and/or amino acidsin cells, and in preventing suboptimal level of carbohydrate catabolism.The improvements are achieved firstly by improving mitochondrial ATPproduction, and secondly by improving antioxidative state of the cells,e.g. by hindering excessive radical oxygen species (ROS) formation fromoxidative phosphorylation (OXPHOS), and thirdly by improving theredox-state, i.e. by increasing cellular capacity to adjust cytosolicNAD+/NADH-ratio in timely manner when needed and, fourthly by theincrease of mitochondrial activity and enhanced IMM membrane potentialsand their control.

The use of DGA enhances mitochondrial aerobic metabolism in cells andbiogenesis of new mitochondria with alleviating effect towardsnon-communicable mitochondrial diseases. The invention also relates tothe use of said compounds in mitochondrial coordination of optimalNAD(P)+/NAD(P)H-ratios in cytosol, cells, tissues/organs and wholephysiological systems, e.g. cardiovascular and/or central nervoussystems.

Further, the invention relates to the use of said compounds in enhancinganaplerotic and anabolic processes like glyceroneogenesis, proteinsynthesis, and pentose phosphate pathway producing ribose-5-phosphate,the precursor of nucleobases adenine and guanine. Directly related tothe above the use of DGA relates also to reducing the formation ofreactive oxygen species (ROS) with alleviating effect towardsnon-communicable diseases related to oxidative damage to DNA, e.g.slowly advancing degenerative diseases and cancer.

The invention is directed to giving cells tools to combat deterioratingredox state during metabolic stress and physical exercise.Simultaneously it enhances antioxidative state of the cells, enhancesmetabolic flux, and also balances ETS and ATP production. In somephysiological states the invention also opens up temporarily apossibility for faster and more sustainable, but also somewhat lessefficient, ATP/energy production by the ETS compared to “full” ETSstarting from complex I, i.e. GP-shuttles.

Based on clinical gene expression and other studies (Examples 2.2, 2.3,and 2.4) it seems that the very short term and longer term effects ofthe use of DGA on aerobic metabolism differ slightly. This due to thefact that the use of DGA causes also structural improvements in energymetabolism that cannot be realized in acute administration becausestructural changes take time, even though they seem to be surprisinglyfast also.

In the short run the biggest and almost immediate improvement achievedby the use of DGA is the increasing cellular capacity to adjustcytosolic NAD+/NADH-ratio. This is due to signal effect (See FIG. 1bbelow) and by directly providing substrates through glyceroneogenesis tothe GP-shuttles. Immediately thereafter and also due to improvedcytosolic NAD⁺/NADH-ratio also MA-shuttles are activated. Aerobicmetabolism causes ROS production. The use of DGA can efficiently fightagainst excessive ROS amount by activating Nrf2/ARE pathways (see FIG.2).

In the longer run (meaning from already 2-4 days onwards) the mainenergy metabolism related effect of the use of DGA is the increase inbeta oxidation, i.e. enhancement of the metabolism (catabolism) of fatsas the energy source. Triglyceride transport through blood circulationis increased, which is a sign that the heart, skeletal muscles and someother tissues have increased their use of fatty acids (FAs) as theirenergy source. It is also reasonable to expect that the de novosynthesis of FAs for the use of cellular energy metabolism is increased.Formed medium chain FAs might possess also other health effects besideson top increased aerobic metabolism and related enhanced ROS control.

It was now surprisingly found that the use of a compound fromD-glycerate group i.e. D-glyceric acid, DL-glyceric acid (DLGA),L-glyceric acid (LGA) and/or hydroxypyruvate enhance mitochondrial ATPproduction, and simultaneously reduce excessive radical oxygen species(ROS) formation from oxidative phosphorylation (OXPHOS), and furthermore can increase cellular capacity to adjust cytosolic NAD⁺/NADH-ratioin timely manner when needed. (see Example 2.3.2 for relevant geneexpression changes, and Example 2.3.3 for significant changes in bloodsubstrate concentrations due to the use of DGA, and FIG. 6-11 fordecline in ROS and increase in ATP use)

Unless otherwise specified, the terms, which are used in thespecification and in the claims, have the meanings commonly used in thefield of biochemistry, particularly in the field of metabolic orexercise/sports related studies.

The term “D-glycerate group” includes the compounds D-glyceric acid,DL-glyceric acid, L-glyceric acid and hydroxypyruvate and their saltsand esters and derivatives.

The term “subject in need” refers to humans and animals. The compositionof the present invention is useful for enhancing metabolism in subjectsin need. The composition is suitable for use in humans. The compositionis also suitable for animals.

DGA is a weakly acid compound that is readily soluble in water andalcohol and can be prepared e.g. by oxidation of glycerol. DGA can beliberated from its commercially available calcium salt form by simpletreatment with dilute hydrochloric acid. Being an organic acid, DGA isalso capable of forming esters. DGA can be liberated from its esters,for instance, by esterase enzymes. In the human body, these enzymes arepresent in the wall of small intestine where they split esterifiednutrients into a form that can be adsorbed from the digestive tract. DGAis typically not directly involved in the normal growth, development orreproduction of an adult organism. Unlike its phosphorylated forms(phosphoglycerates) DGA is not produced in bigger amounts during normalsugar catabolism in the human body. Only very small amounts of DGA havebeen found in the body (Hoffman et al. 1993). LGA is biologicallyrelatively inactive enantiomer. Nevertheless it can be converted intoHPA in the body and thus can possess beneficial properties of theinnovation. DLGA is racemic form of DGA and LGA. HPA is oxidized form ofDGA. HPA can also be formed from L-serine and pyruvate. In this reactionone alanine and one HPA molecule is formed.

The invention is described in detail below with reference to theFigures.

FIG. 1a describes mitochondrial metabolism. Final catabolism ofcarbohydrates and fatty acids (and carbohydrate parts of amino acids)into carbon dioxide (CO₂) and water (H₂O) occur in the mitochondrial TCAand ETS. FIG. 1 furthermore depicts malate-aspartate (MA)-shuttle andglycerol phosphate-shuttle (GP-shuttle). These shuttle mechanisms areneeded because IMM is impermeable for NADH and NAD⁺. MA-shuttlestransport NAD⁺ from the matrix to the inter membrane space(IMS)/cytosol. GP-shuttles are located on the outer side of IMM anddonate electrons from cytosolic NADH directly to the ubiquinol (QH2) inthe ETS and simultaneously increase cytosolic NAD⁺-pool by one NAD⁺. Ontop of being electron carrier in the ETS ubiquinol is a potentlipophilic antioxidant capable of regenerating other antioxidants suchas alpha tocopherol (Vitamin E) and ascorbate (Vitamin C).

MA-shuttles are the predominant shuttle mechanism in mammalian cells.They work basically on continuous, demand driven basis to keep cytosolicNAD⁺ at sufficient levels to allow e.g. normal flow of glycolysis.Mechanism of action of MA-shuttles is relatively slow due to complicatedmechanism of action. By increasing pyruvate concentration in the cellsthe use of DGA can enhance functioning of MA-shuttles. The use of DGAactivates also GP-shuttles that consist of membrane bound mitochondrialglycerol phosphate dehydrogenase (GPD2) and cytosolic glycerol phosphatedehydrogenase (GPD1). GDP2 oxidizes glycerol phosphate (G-3-P) intodihydroxyacetone phosphate (DHAP) and simultaneously reduces one flavindinucleotide FAD into FADH2. GDP2 further oxidizes created FADH2 back toFAD by simultaneous reduction of ubiquinone (Q) to ubiquinol (QH2) in ahydrophobic reaction. In IMS/cytosol GPD1 reduces DHAP back to GP3 andsimultaneously oxidizes cytosolic NADH+H⁺ into NAD⁺.

GP-shuttle is irreversible. MA-shuttle is partially irreversible, i.e.the aspartate (ASP) side with glutamate functions only to one directionbut malate (MAL) can be interchanged with alfa-ketoglutarate (aKG) orwith phosphate also out from the matrix (FIG. 3a ). This partiallyinverted MA-shuttle can export mitochondrial NADH equivalents out fromthe matrix and thus reduce the pressure towards complex I to oxidizeNADH inside the matrix. This happen e.g. in gluco- and glyceroneogenesissituations when oxaloacetate (OAA) from the matrix is exported viamalate to the cytosol for re-conversion back to OAA and further tophosphoenolpyruvate (PEP) or to aspartate-asparagine route to proteinsynthesis (see FIG. 1b and FIG. 3a ).

The use of DGA promotes especially glyceroneogenesis (shown e.g. by theincrease in PGC-1a, in blood triglycerides and pyruvate). The rise inMA-shuttle intermediates arises from increased pyruvate formation(Example 2.3.3). In some situations, e.g. in intensive exerciseglycolysis produces excessive amounts of NADH in short time orglycolysis is inhibited by lack of NAD⁺. In these situations it isbeneficial 1) to rapidly oxidize part of the NADH outside of themitochondrial matrix like GP-shuttles do, and 2) that also MA-shuttlesfunction efficiently. The present Invention provides that GP-shuttlemechanisms can possibly exist also in other cell membranes than inmitochondrial ones.

FIG. 1b describes signaling and related metabolic pathways. As seen inExamples 2, 3.2, and 5, DGA administration can increase aerobicmetabolism significantly and the effect is almost immediate.Furthermore, this effect is sustained also in the longer run (4 days, 3weeks and even for 8 weeks). This is very likely due to some enzymeactivation in correct location of the cytosol and/or IMS that gives astrong signal for cells to activate aerobic metabolism and related vastset of health benefitting effects (see FIG. 2). Without clear signalingeffect it is impossible to explain how relatively small DGA (or HPA)administration could induce strong ETS gene activation, antioxidantdefense activation, increase in triglyceride synthesis and simultaneousincrease in pyruvate and decrease in lactate levels. Further DGA and HPAcan directly complement the activation of aerobic metabolism becausethey themselves provide right substrates for the initiation of e.g.glyceroneogenesis, and the TCA cycle via pyruvate increase, like seen inFIG. 1 b.

Most probable candidate for the location dependent signaling is theactivation of GLYCTK1 and/or GLYCTK2 enzymes in the main direction ofDGA and HPA metabolism. High and prolonged ATP demand, like seen e.g. inendurance exercise, likely activates GLYCTK1 and/or GLYCTK2 genes (thatcan yield ATP). That is likely also why DGA and/or HPA administration isable to activate mitochondrial aerobic energy metabolism, including betaoxidation. At the same time, it should be noted that complex cellsignaling network can also require initial activation or evendeactivation of some other related enzymes or pathways, e.g. D-glyceratedehydrogenase (DGDH) and glyoxylate/hydroxypyruvate reductase (GRHPR).Additionally aldehyde oxidase (AOX1) can be involved in this cellularprocess. (AOX1 is not presented in the graph but it is activated as partof the Nrf2/ARE system, see Example 2.3.2 and FIG. 2.) Activation caninvolve also some relevant candidate from vast aldehyde dehydrogenase(ALDH) family, and/or increased peroxisomal or ER gene activity.Whatever the signal mechanism is, the use of the use of DGA initiatessimilar, very health promoting mechanisms that can be associated withlong term aerobic exercises. But additionally, because of formed extrapyruvate is not consumed, there is clearly even more benefits to begained for vast amount of therapy areas then simply from initiating longterm exercises. Also therapeutic use of e.g. HO-1 (Nrf2/ARE) up and downregulation gives numerous therapeutic possibilities for the use of DGA(more on HO-1 in FIG. 2 and gene expression studies, Examples 1.3 and2.3.2).

Presented feedback mechanism between DGA and HPA likely further enhancesthe positive activation effects of various NADH and NADPH dependentoxidation-reduction-reactions by the use of DGA and/or HPA. DGA-HPA-loopis due to the fact that DGDH and GRHPR can utilize both NADH and NADPHas co-substrates in oxidation-reduction-reactions and even to bothdirections in FIG. 1 b. Favored reaction directions of these enzymes areas mentioned in the names of the enzymes. DGDH favors NAD+ as aco-substrate and GRHPR favors NADPH. From the “DGA-HPA loop” cells getadditional tools to balance NAD⁺/NADH-ratio (energy metabolism)—andimportantly also NADPH/NADP+-ratio (antioxidant defense andanti-inflammatory control) (see FIG. 2 and FIG. 4). DGA-HPA loop islikely an important factor in observed efficacy of the use of DGA and/orHPA in longer term administration, but the use of DGA and/or HPA doesnot provide (or need) that DGA and/or HPA carbon skeletons shuttle onaverage multiple times between DGA and HPA.

Direction from DGA towards D-glyceraldehyde (D-GALD) is not favored dueto the fact that ALDH enzyme activity clearly favors direction towardsDGA. Additionally ALDH enzymes are typically most active inmitochondrial matrix, i.e. not in cytosol/IMS.

From upper right hand corner of FIG. 1b it can be seen that generatedG-3-P that can be used in packaging of fatty acids (FAs) in totriacylglycerides (trigys). Trigys are used in intracellular storage andextra cellular transportation of FAs into tissues. When need ariseslipase enzymes liberate FAs to be used in beta oxidation andsimultaneously liberated free glycerol can be phosphorylated by glycerolkinase back to glycerol phosphate (G-3-P). G-3-P can be also used in thebiosynthesis of phospholipids in the endoplasmic reticulum (ER). ERoften has a close physical interplay with mitochondria in cells. Gluco-and glyceroneogenesis requires generally more energy (ATP, GTP and UTP)than glycolysis produces.

From lower right hand corner of FIG. 1b it can be seen that part of theHPA can be converted with alanine (ALA) into pyruvate (PYR) and L-serine(SER). This transaminase reaction materializes typically in peroxisomesusing transaminase enzymes (AGXT1 and AGXT2). In some tissues thistransaminase reaction can happen also in the mitochondrial matrix andpossibly also in the cytosol/IMS. Peroxisomes generate ROS. In theperoxisomes L-serine with glyoxylate (GLYO) can be converted back to HPAand glycine (GLY) the simplest amino acid. Pyruvate can be alsoconverted back to alanine in cytosolic or mitochondrial transaminasereaction with glutamate (not in the graph). In a situation with excessALA and GLYO this HPA-SER-reaction series can convert excess ALA andGLYO into pyruvate and glycine. Transporting mechanisms of alanine andglyoxylate into peroxisomes exist. There are some reports in theliterature that very high HPA amounts can be harmful for e.g. glialcells in cell cultures. This is likely due to excessive conversion ofHPA and glycine into glyoxylate and L-Serine. Decreased amounts ofglycine can be harmful for the functioning of the CNS. This view on thepossible cause for HPA toxicity in the CNS tissues has not been reportedin the literature before. The amounts of DGA and HPA that are needed inthe present invention are clearly lower than possibly toxic amounts ofHPA. FIG. 2 describes induced master regulatory genes and pathways.Virtuous cycle formed by the use of DGA of enhanced energy metabolismand endogenous antioxidant defenses with increased mitochondrialactivity and mitochondrial biogenesis and increased pyruvateconcentration with less ER stress creates wide range of specific andalso pleiotropic therapeutic effects that can alleviate, prevent or evenheal basically all non-communicable diseases related to dysfunction inenergy metabolism, increased ROS formation, and/or unregulatedanti-inflammatory disorders.

Additionally the activation of pentose phosphate pathway increases eventhe production of nucleobases adenine and guanine thus helping the denovo formation of important biological molecules related to DNAformation and energy metabolism. We have shown that even short 12 hourclinical, in vivo administration of DGA increases gene expression ofGPD2 in peripheral leukocytes. Genes related to complex III (MT-CYB) andIV (COX1) of the ETS increased statistically very significantly after 4day administration implying that aerobic energy metabolism and nuclearrespiratory factor 1 (NRF1) were activated. The use of DGA can activatecellular aerobic energy metabolism also the expression of PGC-1aincreased statistically significantly in leukocytes already after 4 dayadministration of DGA (in vivo), and in hepatocytes in 2 days (in vitro)compared to zero control. PGC-1a increases mitochondrial biogenesis byactivating vast amount of aerobic energy production, i.e. oxidativephosphorylation, related genes. To confirm the case of increased aerobicmitochondrial metabolism by the use of DGA, plasma lactate has beenshown to decrease by more than 30%. This is a very remarkable and strongindication of enhance oxidative capacity of cells. PGC-1a has also beenassociated with increased beta oxidation and glyceroneogenesis.Furthermore PGC-1a has been associated with reduced ER stress.PERK(=RNA-dependent protein kinase (PKR)-like ER kinase) is a key ERstress sensor of the unfolded protein response, is uniquely enriched atthe mitochondria-associated ER membranes. Activation of PERK in unfoldedprotein response situation is necessary and sufficient condition onNrf2/Keap1 dissociation and subsequent nuclear import.

Heme oxygenase-1 (HO-1) expression is strongly activated after 4 dayswith higher doses of DGA but notably smaller doses of DGA can also downregulate the activity of HO-1 after 4 day administration (see Example2.1) and also in 12 h after first doses (Example 2.3.2).

Use of DGA increases blood pyruvate levels 20-25%. In literatureincreased pyruvate concentrations have been inter alia shown toalleviate and, when needed, also activating inflammatory responsesmediated by NF-kB, a pro-inflammatory transcription factor.

The use of DGA can efficiently enhance the in vivo activity level ofantioxidant and anti-inflammatory defenses of the cells, andsimultaneously improve oxidative and inflammatory status of cells,tissues, organs and whole physiological systems. The improvement instatus is followed from enhanced endogenous energy production andnotably increased supply of exogenous energy fuel for certain tissuesespecially in brains and elsewhere in the CNS in the form of pyruvate.The use of DGA provides even several adjacent and independent mechanismsthat produce and/or activates pleiotropic therapeutic events thusensuring that some positive therapeutic effect will be materialized inall subjects in need.

Rate limiting enzyme of the pentose phosphate pathway (G6PD) isactivated rapidly i.e. in 12 h after first administration. Theexpression of G6PD remains at high levels compared to the zero controlalso after 4 days. HO-1 expression is strongly associated with Nrf2 aswell as G6PD gene also. In the nucleus Nrf2 binds to antioxidantresponse element (ARE) that initiate antioxidative genes, e.g. G6PD andHo-1, transcription, and also many mitochondrial transcription factors.

Like PGC-1a, also Nrf2/ARE promotes mitochondrial biogenesis. Nrf2/AREactivates antioxidant defenses of the cells by e.g. activating genesrelated to mitochondrial NADPH-shuttles including malic enzyme andcytosolic iso-citrate dehydrogenase (IDH1). These NADPH-dependentchannels also play a pivotal role in activated antioxidant defenses bygenerating NADPH form NADP⁺. Clear in vitro results with human primaryhepatocytes showing that DGA administration can sharply reduce ROSlevels further supports the relationship that DGA administrationsignificantly increases antioxidant Nrf2/ARE-pathway. Separate in vitroresults from rat cortical neurons show increased mitochondria biogenesisafter 4 day administration of DGA. Positive test results in peripheralleukocytes, neurons and hepatocytes support the idea that both PGC-1aand Nrf2/ARE are activated by DGA in all cell types that usemitochondrial energy metabolism as their primary source of energy.

FIG. 3a relates to fasting state. Fasting and resting state, e.g. duringthe night and DGA or HPA administration before going to bed, is veryfavorable for beneficial anabolic and anaplerotic actions of the DGA use(on top of the activation of Nrf2 and PGC-1a/NRF1 pathways). In fastingDGA administration increases 1) glyceroneogenesis that providessubstrates to the GP-shuttle (and seen as an increase in GP-shuttleactivity in gene expression), 2) beta oxidation that is inter aliaindicated by significant increase in glycerol and pyruvate levels (seeExamples 5 and 2.3.3), and 3) activity of MA-shuttles and the TCA (dueto increase in pyruvate). In the longer run more permanent increase inbeta oxidation leads to enhanced intracellular triglycerides formationand lipase activity in tissues e.g. in muscle cells (like in example 5).In the short run increased demand for triglycerides in muscles isprovided mostly by the liver, and seen as a temporary increase in bloodtriglycerides levels (Examples 2.1., 2.2 and 2.3.1).

DGA can facilitate enhanced NADH oxidization into NAD⁺ and thus e.g.lactate (LAC) conversion into pyruvate (PYR). In Feedback effect fromHPA back to DGA can activate e.g. malic enzyme (ME) that converts malate(MAL) into pyruvate. ME on the other hand is related to Nrf2/ARE nucleartranscription factor. Formed excess amount of pyruvate can be rapidlyused as redox-regulator (reaction back to lactate), or in energyproduction through TCA and/or in fatty acid synthesis and/orgluco-/glyceroneogenesis, and further even into protein synthesis viaOAA. Cytosolic OAA can be used in gluco-/glyceroneogenesis or betransaminated with glutamate (GLUT) into alfa-ketoglutarate (aKG) andaspartate (ASP). Aspartate with glutamine and ATP can be furtherconverted into asparagine and glutamate and AMP+PPi. (Glutamine isformed from glutamate and ammonia using one ATP into ADP+Pi, not in thegraph.) Energy needed for these anabolic reactions in this fasting stateis provided typically by beta oxidation. As an example related toincreased glyceroneogenesis, healthy and/or trained skeletal musclecells start to form increased amounts of triglycerides close tomitochondria from glycerol phosphate and fatty acids as an efficientenergy source for future mitochondrial beta oxidation. On the proteinsynthesis side more energy needing asparagine has been reported to be asuperior building block for functioning proteins compared to e.g.glutamine. All in all, enhanced energy production by the use of DGAfacilitates healthy metabolism.

Dotted line implicating pyruvate net export from the cells through MCTs(monocarboxylate transporters) happens in bigger scale only inglycolytic cells that can't further use pyruvate in their energymetabolism. These cells include e.g. red blood cells (RBC), glycolyticoligodendrocytes and some other glial cells, and additionally glycolyticmyocytes. Those cells have totally or partially lost their mitochondrialactivity and have been specialized in some important support role forother cells and tissues, like oxygen transport for RBCs or support ofaxonal integrity in the CNS. By being able to provide NAD+ and NADPHsimultaneously and repeatedly the DGA-HPA -loop can enhance glycolysisand thus ATP and pyruvate production of also glycolytic cells, andsimultaneously enhance the antioxidant defense of these those cells. Theeffect of the use of DGA to energize neuronal axons possesses positiveeffect in preventing and alleviating e.g. neurodegenerative diseases. Itis possible that headache, the only withdrawal effect seen thus far inExamples 2.1, 2.2 and 2.3, is a cause of neurons getting used to betterenergetic environment with the use of DGA. Outside of theblood-brain-barrier the significant increase of blood pyruvateconcentration by the use of DGA implies significant average increase ofintracellular pyruvate concentration due to automatic balancing ofexcessive concentration gradient over plasma membrane by themonocarboxylate transporters (MCTs). In literature administration ofpyruvate in various forms e.g. as pyruvate salt or as ethyl pyruvate hasbeen associated with several beneficial effects including an increase inactivity of the Nrf2/ARE mediated pathways. As shown by the Example 2.1and 2.2 the use of DGA can bring similar positive therapeutic effects invivo and with relatively low DGA administration.

Worth noticing also is that in cerebrospinal fluid (CSF) of healthyindividuals the concentration of DGA is clearly higher than in blood(Hoffman et al) implying that DGA has a role in normal healthy CNSmetabolism. Clear concentration gradient across the BBB, implies thatthere exists some kind of monocarboxylate transporting mechanism acrossthe BBB for DGA that notably prevents passive diffusion from CSF to theblood.

FIG. 3b relates to fed state. In fed state blood glucose (GLUC) levelstypically increase and in healthy individuals blood insulin levels rise.Insulin initiates complex process in which inter alia certain glucosetransporters (GLUT4) are transported to the cell surface. GLUT4 areinsulin sensitive glucose transporters e.g. in skeletal muscle, hearthand adipose tissue and to lesser extend also in the CNS. Thetransportation of GLUT4 needs energy. Insulin, on the other hand, issynthesized in the pancreas within the so called β-cells. Interestinglyglucose stimulated insulin secretion (GSIS) is stimulated bymitochondrial citrate iso-citrate carrier (CIC) in β-cells, as well asATP/ADP ratio and NADPH/NADP⁺ ratio of the β-cells. The use of DGAfacilitates all mentioned GSIS stimulators and thus it is not surprisingthat the insulin levels in blood seem to increase in fed state by theuse of the use of DGA. Accordingly glucose intake by the cellsincreases.

An additional explanation to increased glucose intake effect of the useof DGA could be the increased production capacity of ATP by the cells.ATP (and UTP) facilitates glucose intake also by converting GLUC intoG-6-P or glycogen and thus prolonging inflow of glucose from blood intocells.

Elevated cellular pyruvate (PYR) concentration increases Acetyl Coalevel and further more citrate concentration. Increased export ofcitrate and its cytosolic conversion into Acetyl Coa and OAA is animportant step in fatty acid (FA) synthesis and also for so calledmevalonate pathway. Increased Acetyl Coa levels can additionallyactivate also pentose phosphate pathway that can use imported andphosphorylated glucose (G-6-P) for ribose-5-phosphate (R-5-P) synthesis,and also to provide efficient enhancement of cytosolic NADPH/NADP⁺ ratioin the cells.

Increased aerobic ATP production increases ROS production that is likelyone factor activating Nrf2/ARE antioxidant defense mechanisms of thecells by the use of DGA. In literature it is reported that Nrf2/ARE canactivate glutathione (GSH) as well as mitochondrial superoxide dismutase(SOD2) production in the cells, and thus facilitate the formation ofvery efficient endogenous antioxidant defense for cells. As has beenmentioned earlier the use of the use of DGA can facilitate ROSscavenging very efficiently compared to zero control in human primaryhepatocytes (Example 1.1 and 1.2). The use of DGA provides antioxidantprotection also during fasting state. Target use of the use of DGA isagainst cancer, to almost all degenerative diseases, many autoinflammatory and autoimmune diseases, diabetes, cardiovascular diseasesand various myo- and neuropathies related to aerobic energy metabolismand mitochondrial disorders.

FIG. 3c describes neuronal protection in NMDA stimulation (excitotoxicinsult) disclosed by Flint Beal M., (Trends in Neuroscience 23: 279-330,2000): “A severe excitotoxic insult (Case I) results in cell death bynecrosis, whereas a mild excitotoxic insult (Case II) results inapoptosis. After a severe insult (such as ischemia), there is a largeincrease in glutamate activation of NMDA receptors, an increase inintracellular Ca2+ concentrations, activation of nitric oxide synthase(NOS), and increased mitochondrial Ca2+and superoxide generationfollowed by the formation of ONOO—. This sequence results in damage tocellular macromolecules including DNA . . . . A mild excitotoxic insultcan occur due either to an abnormality in an excitotoxicity amino acidreceptor, allowing more Ca2+ flux, or to impaired functioning of otherionic channels or of energy production, which may allow thevoltage-dependent NMDA receptor to be activated by ambientconcentrations of glutamate. This event can then lead to increasedmitochondrial Ca2+ and free radical production, yet relatively preservedATP generation.”

In neurons, that possess very high oxygen and ATP need, lactate is oftenproduced in so called aerobic glycolysis in cytosol as its end product,and then it is shuttled into the matrix through MCT (see FIG. 3b ). Inthe matrix lactate is converted back to pyruvate. This mechanismprovides additional shuttle mechanism for shuttling NAD⁺s into cytosolfrom the ETS (Complex I and/or the ETS) besides MA- and GP-shuttles.Neurons need excess amounts of NAD⁺-shuttling capacity form mitochondriaprobably because they rely mainly on glycolysis in their energyproduction. The use of DGA can enhance NAD⁺ shuttling capacity and thusit can alleviate or even heal especially dysfunctions related to theenergy metabolism in the CNS (e.g. basically all neurodegenerativediseases but also epilepsy, bi-polar disorder etc.).

The use of DGA has been shown in vitro to protect rat cortical neuronsagainst excitotoxic insult caused by NMDA stimulation (see Example 3.1).Excitotoxicity may be involved in spinal cord injury, stroke, traumaticbrain injury, hearing loss (through noise overexposure or ototoxicity)and in neurodegenerative diseases of the central nervous system (CNS)such as multiple sclerosis, Alzheimer's disease, amyotrophic lateralsclerosis (ALS), Parkinson's disease, over-rapid benzodiazepinewithdrawal, and also Huntington's disease. One explanation for theprotection against excitotoxic insult is increased ATP production by theuse of DGA. ATP facilitates calcium storage into ER and also its effluxout of the neuron by ATP dependent plasma membrane Ca²⁺ ATPase.Increased ATP production is important facilitator in neutralizing ofexcess calcium levels but Ca²⁺ ATPase is not very fast exporter ofcalcium and thus neurons need also so called Na⁺/Ca²⁺ exchangers forrapid clearance of excessive cytosolic calcium that can cause celldeath. On top of rapid ATP production neurons need also glial cells intheir adjustment and protection against external and internal stresses.According to current understanding close interplay between astrocytesand neurons in the CNS give neurons the protection by the Nrf2/AREsystem e.g. against NOS induced cell damage (see FIG. 4 and Example 3.1for further information). Also increased mitochondrial biogenesis (shownin Example 3.2) can facilitate calcium regulation and protect againstexcitotoxic insult.

In the CNS increased expression of PGC-1a can also limit beta-amyloidformation that is considered one major reason for Alzheimer's disease(AD). In the literature it is shown that peripheral leukocytes can givegood indication on e.g. pathological development in AD. In vivo clinicalexperiments have indirectly shown that the use of DGA can efficientlyalleviate normal metabolic challenges in the CNS in healthy volunteers,because mild but clear negative effects like headache have been reportedafter stopping the use of the use of DGA. DGA probably cannot freelycross the blood brain barrier but instead uses similar transportingmechanisms as pyruvate and other small carboxylic acids. Interestinglyit has been shown in literature that pyruvate can alleviate glucosedeprivation induced increase in beta amyloid formation in the braintissue. The use of DGA can increase pyruvate concentration in blood infasting and fed states.

In vitro neuronal cell culture studies are often done without ampleamounts glial cells like astrocytes in the culture. This means that e.g.Nrf2/ARE antioxidant protection mechanisms provided almost solely byastrocytes in the brains are often missing to a large extent from thesestudy set ups. Astrocytes have been shown also to provide lactate andpyruvate for neuronal ATP production by oxidative phosphorylation.

FIG. 3d depicts one mechanism of action of the DGA and/or HPA use in redblood cells (RBC or erythrocytes). Mature RBC lack mitochondria, nucleusand basically all other cell organelles that are replaced by hemoglobinmolecules. RBCs' main and maybe only major role in an organism is totransport oxygen into tissues and export CO₂ with hemoglobin molecules.RBCs have a life span of only some 120 days and thus new ones are beingproduced some millions every second in adult humans. RBCs produce theirATP energy via glycolysis. Formed NADH molecules can't be used forenergy production in the ETS and they are mainly used and converted backto NAD⁺ in NADH dependent methemoglobin reductase pathway that convertsmethemoglobin into hemoglobin by oxidizing its iron molecule into Fe²⁺.This critical conversion makes it possible for hemoglobin to bindoxygen. Important to notice that also NADPH can be used in differentkind of methemoglobin reductase reactions (also presented in FIG. 3d ).Due to lack of mitochondria, it is especially important to keep NAD+ andNADPH producing mechanisms active and functioning in RBCs. NAD+ isneeded for ATP production and NADPH for antioxidant defenses. Majorsource of NADPH molecules in RBC is the pentose phosphate pathway. Inthe literature it is shown that both enzymes DGDH and GRHPR are activein RBCs. One NADPH (or NADH) can be produced when moving from DGA toHPA, and NAD⁺ (NADP⁺) when moving back to DGA from HPA. The inventionactivates DGA-HPA-loop reactions in the RBCs and makes balancing ofNAD^(+l /NADH and NADPH/NADP) ⁺-ratios more efficient. The use of DGAhelps RBCs' energy production, antioxidant defense, and hemoglobinintegrity (increased viability of RBC is indicated by observed declinein serum LDH in Examples 2.1 and 2.3.1). By enhancing the ATP productionof the RBCs and their NAD⁺/(NADH+H⁺)-ratios (=increase in intracellularpH), the invention can increase 2,3-BPG content in the erythrocytes insubjects in need. Importantly 2,3-BPG allosterically increases oxygenreleasing capacity of oxygen, this effect can be significant in tissuesdeprived of oxygen. By enhancing RBC oxygen releasing capacity theinvention can likely alleviate hypertension and possibly congestiveheart failure and similar disorders. By reducing oxidativedamage/increasing NADPH levels, the invention can possibly alleviatesymptoms related to G6PD and 6PGD (6-Phosphogluconate dehydrogenase)deficiencies. Because of lack of peroxisomes and mitochondria, the mainway out from the DGA-HPA loop (besides direct efflux through plasmamembrane) is eventually moving towards glycolysis and towards pyruvate.RBC pyruvate production can increase also due to influx of lactate intoRBCs (that is a well-documented fact) and its conversion towardspyruvate. By enhancing NAD⁺ formation the invention can increasereversed LDH activity of RBCs and efflux of pyruvate into blood stream(as the results of increased blood pyruvate concentration indicate,Example 2.3.3).

FIG. 4 describes NADPH− and Nrf2/ARE related pathways. Most of theseNADPH dependent pathways and genes are also directly or indirectlyrelated to cellular antioxidant defenses, i.e. Nrf2/ARE related genesand pathways. The relationship can be e.g. like presented in FIG. 3bwhere PPP provides NADPH for reducing (activating) glutathione. Ingeneral Nrf2 regulated genes can be divided into three categories: (1)antioxidants, (2) anti-inflammatory, and (3) genes related tomitochondrial biogenesis/protection.

In the FIG. 4 there are also some enzymes and reactions that have notyet been directly tested but instead are logically induced by showneffects of the use of DGA, e.g. glucose-6-phosphate dehydrogenase (G6PD)is the rate limiting step of pentose phosphate pathway (PPP) and thus itnaturally activates next enzymes in the pathway e.g. 6PGD(6-Phosphogluconate dehydrogenase). Furthermore PPP generates a lot ofNADPH that can activate or at least be used as a co-substrate in most ofthe presented reactions. Other abbreviations in the graph:ribose-5-phosphate (R-5-P), β-5-phosphorybosylamine (5-PRA), L-arginine(L-Arg.), NO=nitric oxide, CO=carbon monoxide, Fe=iron, andDHA=dehydroascorbate. For eNOS and iNOS and their relation withHO-1/Nrf2 see below.

Pentose phosphate pathway (PPP) is up regulated relatively fast alreadyin 12 hours in healthy volunteers. The expression of G6PD the ratelimiting enzyme of this important pathway increased statisticallysignificantly in peripheral leukocytes. The reason for this activationcan be increased ROS production from aerobic mitochondrial energyproduction (increase in GPD2, in beta oxidation (acetyl CoA up), andeventually in PGC-1a). ROS scavenging needs NADPH that PPP can supply.Increased ROS production activates also Nrf2 pathway. Nrf2 translocationfrom cytosol into nucleus activate AREs(=antioxidant response elements)of specific antioxidant defense related genes, like HO-1. The use of DGAcan activate HO-1 very strongly, indicating that it can efficientlyactivate Nrf2 pathway. Also increase in blood bilirubin concentrationshave been observed simultaneously after 4.5 day administration of theDGA confirming enzymatic action at substrate level (example 5.3.1 and5.3.2). Importantly, lower administration of the DGA has alsoconsistently decreased bilirubin levels in healthy andexercising(=mitochondrial metabolism using) volunteers. The use of DGAis planned also for the longer term prevention of diseases and healthpromotion, and thus it is important that unnecessary activation ofcellular defense mechanisms is not turned on all the time. In factexamples 1.1 and 2.1 show that most of the time Nrf2 is down regulatedby the use of DGA in humans. Reduced normal ROS levels and simultaneousalert defense systems (with ample ATP energy supply) are the key tolonger term health and extended life span.

It is well known that HO-1 expression and Nrf2/ARE pathway correlatepositively. Nitric oxide synthase (NOS) family inducible NOS (iNOS),endothelia NOS (eNOS) and neuronal NOS on the other hand seem tocorrelate clearly negatively with Nrf2 expression as well as also the NOproduction does, e.g. Nrf2 activation (in astrocytes) can protectneuronal cells against excessive NO generation in excitotoxity model(see FIG. 3c ). Important exception in NO production respect seems to beendothelial cells in which very surprisingly NO production is able toremain at high levels even though Nrf2 clearly depresses eNOSexpression. Possible explanation being simultaneous increase in HO-1expression. (Source: Heiss et al., 3 Biol Chem. 2009 Nov. 13;284(46):31579-86, “Nrf2 Contributes to keep eNOS in the coupled state”).From previous it follows that HO-1 and NOS expressions often correlatenegatively. This could be a follow up of the facts that 1) the productsCO and NO of reactions are complementary in many signaling tasks, andthat 2) both reactions use a lot of NADPH as a co-substrate (FIG. 4).Tentatively the use of DGA seems to be able to increase blood NO levels,i.e. activate eNOS, in humans with metabolic syndrome thus contributingto lowered blood pressure (Example 2.3.3). We also know that DGA canreduce blood pressure in subjects in need with elevated blood pressure(FIG. 12).

Interestingly in healthy volunteers the use of DGA reduces blood urealevels and as follow up it also reduces blood NO levels (see Example 2.1and Example 2.3.3).

Nrf2 and NADPH production contribute clearly and positively to thereduced glutathione (GSH) levels of the cells. GSH is readily usable incells antioxidant defenses. As stated already above increasedantioxidant defense capacity from the use of DGA is very clearly visiblefrom e.g. Examples 1.1 and 2.1. Finally NADPH can activate ascorbate(vitamin C) to its reduced state from DHA. Vitamin C is needed e.g. incollagen synthesis. This aspect is relevant for several therapeuticareas (see indication areas below).

A composition which is useful in the present invention comprises one ormore compounds selected from the D-glycerate group (D-glyceric acid,DL-glyceric acid, L-glyceric acid, and hydroxypyruvatic acid and saltsand esters thereof). Said compounds are for use in a method of enhancingdirect and indirect mitochondrial metabolism. Said compounds or acomposition comprising one or more of said compounds are also for use ina method of treating or preventing a disease or disorder. The disease ordisorder is such as a cardiovascular disease, metabolic syndrome,disorder associated with metabolism, cancer, overweight, elevated bloodpressure, or aging process of an organism, but is not limited to saiddisorders.

The present invention is useful in the therapy areas selected from thefollowing non-limiting groups. Preferably DGA is used.

Cardiovascular diseases: atherosclerosis, myocardial infarction,cardiomyopathy/congestive heart failure, vascular thrombosis and/orembolism, asthma and chronic obstructive pulmonary disease (COPD), G6PDand 6PGD (6-Phosphogluconate dehydrogenase) deficiencies inRBC/hemolytic anemia, lethal sepsis, lethal hemorrhagic shock, andinfant jaundice.

Elevated blood pressure/hypertension: primary (essential) hypertensionor secondary hypertension, including but non-limited to incidentalhypertension and hypoxic pulmonary hypertension.

Disease or disorder related to metabolic syndrome: diabetes, diabeticneuropathy

Disorder associated with metabolism: mitochondrial DNA depletion andother mitochondrial diseases, Leigh syndrome, epilepsy, bipolardisorder, psychiatric disorders and mood disorders, cerebrovascularaccident, damage from acute head injury, acute or chronic renal failure,acute or chronic liver failure, splenomegaly, acute or chronicpancreatic failure, chronic auto inflammation and autoimmune syndromeand diseases, psoriasis, impairment in collagen synthesis,pre-eclampsia, thyroid disease, chronic fatigue, fibromyalgia.

Overweight

Cancer: cancer subtypes: Basically all types of cancers that are causedby ROS damage to the cell, dysfunctioning mitochondria (e.g. compromisedability for apoptosis) or/and dysfunction of the energy production ofthe cells can be postponed or even prevented. In some cases a processthat endogenously could suppress some tumor is facilitated. In generalby alleviating aging related degeneration of the cells, the presentinvention is useful in reducing the number of malignant cells and/orenhancing their controlled cell death. A skilled person in art is ableto select a cancer subtype that can be postponed, alleviated, preventedor suppressed e.g. from the list in National Cancer Institute of US NIH:www.cancer.gov/cancertopics/types/alphalist

Disease or disorder related to aging of an organism: age related hearingloss, including but not limited to presbyacusis, noise induced hearingimpairment, ototoxic hearing impairment), age related maculadegeneration, glaucoma, optic neuropathy and ischemic optic neuropathy,retinitis pigmentosa, osteoporosis, osteoarthritis, chronicneurodegeneration, amyotrophic lateral sclerosis, Alzheimer's disease,Parkinson's disease, multiple Sclerosis, Huntington's disease, priondisease.

Pyruvate therapy: the use of DGA can increase plasma pyruvate levels by25% (shown in Example 2.3.3). An increase in plasma is a directreflection of similar intracellular pyruvate increase through MCTs. ThusDGA can be used for so called pyruvate therapy (PTh) and even substituteit. In PTh pyruvate is administered orally in salt or ester form. It iswell known that administration of pyruvate salt or ester can alleviate,prevent or even heal many diseases and/or disorders such as lethalsepsis, lethal hemorrhagic shock, Leigh syndrome, COPD and otherinflammatory diseases, mitochondrial DNA depletion and othermitochondrial diseases. Oral DGA calcium salt can significantly increaseendogenous pyruvate production and pyruvate levels in plasma and in thecells; moreover the effect is achieved with very low doses compared toorally administered pyruvate doses needed in PTh.

The composition of the present invention is also useful for enhancinggeneral health and wellbeing of subjects in need.

Furthermore, the composition of the present invention is for use in amethod of enhancing oxygen binding of erythrocytes and releasingcapacity of hemoglobin (due to increased cytosolic pH due to convertingNADH+H⁺ into NAD⁺ that increases the production of2,3-bisphosphoglycerate (see FIG. 3d )). The composition of the presentinvention is also for use in a method of lowering blood pressure inpatients with clearly elevated levels.

In a preferred embodiment of the invention the composition comprises oneor more compounds selected from the group consisting of D-glyceric acid,DL-glyceric acid, L-glyceric acid, hydroxypyruvatic acid and their saltsand esters, as the only active substance or substances.

In another preferred embodiment of invention the composition consists ofone or more compounds selected from the group consisting of D-glycericacid, L-glyceric acid, hydroxypyruvatic acid and their salts and esters,as the sole ingredient or ingredients of said preparation.

A composition of the present invention comprising one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, or hydroxypyruvatic acid or their salt or ester is foruse in a method of enhancing physical training, performance and recoveryfrom exercise.

The compounds of the present invention enhance aerobic and anaerobicproduction of energy and enables cells to recover after physicalexercise. For example acidosis in skeletal muscle tissues can bemoderated by increased redox balancing capacity, i.e. transforming NADH+H⁺ into NAD⁺.

A composition comprises one or more compounds selected from the groupconsisting of D-glyceric acid, L-glyceric acid, hydroxypyruvatic acidand their salts and esters for use as an antioxidant or for use as amedicament having an antioxidant activity.

A composition comprising one or more compounds selected from the groupconsisting of D-glyceric acid, DL-glyceric acid, L-glyceric acid, andhydroxypyruvatic acid and salts and esters thereof for use in a methodof increasing the muscle yield per gram of nutrition and simultaneousdecreasing of fat content of humans and animals, and/or alternatively ina method of decreasing nutrition consumption without losing muscle massof the animals including but limited to live stock (mammals), poultry,and fish.

A composition useful in the present invention may be an oral, topical,parenteral, or inhalable composition for enhancing direct and indirectmitochondrial metabolism comprising one or more compounds selected fromthe group consisting of D-glyceric acid, DL-glyceric acid, L-glycericacid, hydroxypyruvatic acid and their salts and esters. The compositionor compositions for use in the present invention may further comprise apharmaceutically acceptable excipient. Suitable conventional excipientand/or carriers which can be used in the present invention are known bythe skilled person in the art.

The composition may be preparation in the form of a solution, syrup,powder, ointment, capsule, tablet or an inhalable preparation. Thecomposition may be in the form of a solution suitable for parenteraladministration.

The various ingredients and the excipient and/or carrier are mixed andformed into the desired form using conventional techniques. Thecompositions of the present invention may also be formulated with anumber of other compounds. These compounds and substances add to thepalatability or sensory perception of the particles (e.g., flavoringsand colorings) or improve the nutritional value of the particles (e.g.,minerals, vitamins, phytonutrients, antioxidants, etc.).

The composition for use in the present invention may be a part of abeverage, a food product, a functional food, a dietary supplement, or anutritive substance.

Said beverage, food product, functional food, dietary supplement,supplementary food, or nutritive substance may comprise one or moreinert ingredients, especially if it is desirable to limit the number ofcalories added to the diet by the dietary supplement. For example, thedietary supplement of the present invention may also contain optionalingredients including, for example, herbs, vitamins, minerals,enhancers, colorants, sweeteners, flavorants, inert ingredients, and thelike. Such optional ingredients may be either naturally occurring orconcentrated forms.

In an embodiment the beverage, food product, functional food, dietarysupplement, or nutritive substance further comprises vitamins andminerals. In further embodiments, the compositions comprise at least onefood flavoring. In other embodiments, the compositions comprise at leastone synthetic or natural food coloring.

The composition of the present invention may be in the form of a powderor liquid suitable for adding by the consumer or food producer to a foodor beverage. For example, in some embodiments, the dietary supplementcan be administered to an individual in the form of a powder, forinstance to be used by mixing into a beverage or bottled water, or bystirring into a semi-solid food such as a pudding, topping, spread,yoghurt, sauce, puree, cooked cereal, or salad dressing, for instance,or by otherwise adding to a food, such as functional food.

The handling of excess energy from excess nutrition intake is improvedin the present invention. The production of energy from digested food isenhanced. In other words the composition used in the present inventionis useful as a diet medicament.

A packaged pharmaceutical preparation useful in the present inventionmay comprise at least one therapeutically effective dosage formcontaining D-glyceric acid, DL-glyceric acid, L-glyceric acid, orhydroxypyruvatic acid or their salt or ester.

An embodiment of the present invention is a pharmaceutical compositioncomprising an effective amount of one or more compounds selected fromthe group consisting of D-glyceric acid, DL-glyceric acid, L-glycericacid, and hydroxypyruvatic acid and salts and esters thereof for use inmethods according to present invention.

A composition useful in the present invention may be a nutritionalpreparation for enhancing the metabolism of carbohydrates, fat and/oramino acids comprising one or more compounds selected from theD-glycerate group (consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, hydroxypyruvatic acid and their salts and esters).

The present invention is also related to a method of enhancing directand indirect mitochondrial metabolism in a subject comprisingadministering an effective amount of one or more compounds selected fromthe group consisting of D-glyceric acid, DL-glyceric acid, L-glycericacid, hydroxypyruvatic acid and their salts and esters to a subject inneed of enhancing the metabolism of carbohydrates, fats and/or aminoacids.

The present invention also relates to a method of enhancing physicaltraining, performance and recovery from exercise, or reducing radicaloxygen species with antioxidants in a subject comprising administering acomposition comprising an effective amount of one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, and hydroxypyruvatic acid and salts and esters thereofto a subject in need.

The present invention also relates to a method of increasing the muscleyield per gram of nutrition and simultaneous decreasing of fat contentof humans and animals, and/or alternatively in a method of decreasingnutrition consumption without losing muscle mass of the animalsincluding but limited to live stock (mammals), poultry, and fishcomprising administering a composition comprising an effective amount ofone or more compounds selected from the group consisting of D-glycericacid, DL-glyceric acid, L-glyceric acid, and hydroxypyruvatic acid andsalts and esters thereof to a subject in need. An embodiment of themethod comprises administering a pharmaceutical preparation comprisingone or more compounds selected from the group consisting of D-glycericacid, DL-glyceric acid, L-glyceric acid, hydroxypyruvatic acid and theirsalts and esters, and a pharmaceutically acceptable excipient. Anembodiment of the method comprises administering an oral preparation inthe form of a solution, syrup, powder, capsule or tablet.

An embodiment of the method comprises administering one or morecompounds via a parenteral solution and topical medicament.

Another embodiment of the method comprises administering one or morecompounds via a beverage, a food product, a functional food product, adietary supplement, or a nutritive substance.

The composition is administered to a subject in need at a dose effectivein enhancing metabolism. An advantage of the present invention is thatthe administrable dose is small allowing a convenient dosage to subjectsin need. The daily dose in humans may be from 0.1 mg/kg body weight to20 mg/kg body weight, such as 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/kg, preferably from 3 to 5 mg/kgbody weight once or twice a day such as 180 mg-600 mg per day for 60 kgweighing person. In animals the daily dosage per kilogram could be alsohigher.

The composition may be used in a mixture for enhancing metabolism in asubject in need. The composition is useful for use as a therapeuticagent. The amount of antioxidants is increased in the body. Thecomposition of the present invention is useful as e.g. a complement tovitamins and flavonoids. The decrease of ROS and reduction orstabilizing of blood cholesterol is observed (Table 3). This amongseveral other shown factors leads to improvements in patients sufferingfrom a cardiovascular disease.

Also patients suffering from cancer and metabolic syndrome may benefitfrom the use of the compositions of the present invention. Anotherexample of a preferable application is use in weight control andreduction.

A useful application of the present invention is in aging process. Thisis supported by results of in vitro cell studies, wherein significantreduction of the amount of ROS was observed. Also in vivo resultssupport the notion that oxidative stress is reduced (Table 4, bilirubin,urate and LDH), and aerobic energy metabolism is significantly increased(Example 2.3.3). Increased ETS activity and mitochondrial beta oxidationis seen in humans in vivo. Also clear activation of endogenousantioxidant defense mechanisms was seen (Nrf2/ARE pathways).

Glycolysis, Beta Oxidation and Energy Metabolism of the Cells

Fatty acid catabolism involves three stages. The first stage of fattyacid catabolism is beta-oxidation, which occurs in mitochondrial matrix.The second stage is formation of acetyl CoA (FIG. 3a ) and its oxidationto carbon dioxide in TCA. The third stage is electron transfer fromformed electron carries in the ETS to oxygen. Fatty acid oxidation alsooccurs in peroxisomes, when the fatty acid chains are too long to behandled by the mitochondria. However, the oxidation ceases atoctanoyl-CoA. It is believed that very long chain (greater than C-22)fatty acids undergo initial oxidation in peroxisomes which is followedby mitochondrial oxidation.

In glycolysis in the cytosol of eukaryotic cells, phosphorylated glucose(G-6-P) is converted to pyruvate (PYR), with the net formation of twoATPs and the net reduction of two NAD⁺ molecules to NADH+H⁺. ATP isformed by two substrate-level phosphorylation reactions in theconversion of glyceraldehyde 3-phosphate to pyruvate. Pyruvate can enterthe mitochondrial matrix and convert to acetyl CoA, and thereafter toenter citric acid cycle (TCA cycle) that also uses NAD⁺ as a catalyzingoxidative agent.

Both beta oxidation and glycolysis (via pyruvate) produce acetyl CoA forthe TCA. When beta oxidation is active there is ample amount of acetylCoA around. In these situations pyruvate accumulates into the cells andbody (like seen in example 2.3). This is why we can state that anincrease in pyruvate is an indication of increased beta oxidation. Ofcourse we need also other proof for the case of beta oxidation, like anincrease in blood triglycerides and free glycerol from lipase reaction.

Enhancement of PPP by the use of DGA is an indication of increasedcytosolic acetyl CoA levels, but that can be due to both an increase inpyruvate or in beta oxidation. Additionally in example 1.2.1 we haveseen a dose dependent decrease in NAD+/NADH levels indicating that onwhole cell level the metabolic activity has increased, but in there alsowe can't make a distinction on the exact source of the increment,because all energy producing pathways (including also the TCA) convertNAD⁺ into NADH.

Accumulated pyruvate is a fact and it can be also used in anapleroticand anabolic reactions. Depending on the redox -state of the cell it canalso convert to lactate using reduced NADH as a co-enzyme, and producingcytosolic NAD⁺. This ability of pyruvate to produce cytosolic NAD⁺rapidly is an additional, important feature of the use of DGA in e.g.preventing of AGEs formation. In general lactate levels seem to decreasemore than 30% by the use of DGA which is very clear proof on theefficacy.

The rate of glycolysis, beta oxidation and the TCA, which depends on thecell's need for ATP, is controlled by the inhibition and stimulation ofseveral enzymes. This complex regulation coordinates the activities ofthe glycolytic pathway, beta oxidation and the citric acid cycle and canresults in the storage of glucose as glycogen or as fat when ATP isabundant.

Lactate formed in the skeletal muscles can be exported to the liver andconverted there back to pyruvate and further to gluconeogenesis in theliver and later moved back to the muscles as glucose (so called Coricycle).

Mitochondria have a permeable outer membrane and an inner membrane,which is the site of electron transport system and ATP synthase.Pyruvate dehydrogenase, a very large multienzyme complex in themitochondrial matrix converts pyruvate into acetyl CoA and CO2. In eachturn of the citric acid cycle, acetyl CoA condenses with the four-carbonmolecule oxaloacetate to form the six-carbon citrate, which is convertedback to oxaloacetate by a series of reactions that release two moleculesof CO2 and reduce three NAD⁺ into NADH molecules and one FAD into FADH2and further one ubiquinone into ubiquinol and one GDP into GTP molecule(FIG. 1). CO₂ is exhaled or converted to bicarbonate and excreted viaurea cycle to urine.

The NADH generated in the cytosol during glycolysis can be re-oxidizedto NAD⁺ e.g. with the concomitant reduction of NAD⁺ to NADH in themitochondrial matrix, by a set of enzymes and transport proteins thatform shuttle mechanisms through the inner mitochondrial membrane (IM),e.g. so called malate-aspartate-shuttle or glycerol phosphate shuttle(FIG. 1a ).

In the matrix reduced NADH5 from the TCA and from above mentionedshuttle mechanisms are mostly transported to the so called electrontransport system (ETS) located in and on the IM (FIG. 1a ).

In the ETS, electrons from NADH and FADH2 move via a series ofmembrane-bound electron carriers in the inner mitochondrial membrane toO₂, regenerating NAD⁺ and FAD. This stepwise movement of electrons iscoupled to pumping of protons across the inner membrane. The resultingproton-motive force powers ATP synthesis by creating electric gradientover inner membrane of mitochondria and generates most of the ATPresulting from aerobic oxidation of glucose.

ETS consists of several complexes that transport electrons and bumpprotons from the matrix to the inter membrane space (IMS). The endproduct of ETS is water and formed electrochemical gradient can“energize” ADP molecules into ATP inside the matrix in ATP synthasecomplex spanning through the inner membrane. Pumping ATP out of thematrix and ADP into the matrix requires some energy thus consuming partof the electrochemical gradient (FIG. 1a ). Most of the complexes in ETSspan throughout the inner membrane but so called complex II type ofshuttle mechanisms operate only from one side of inner membrane.“Standard” complex II shuttle is located inside the matrix and it formsalso one step in the TCA, i.e. reaction from succinate to fumeratecatalyzed by flavin adenosine dinucleotide (FAD). GP-shuttles areComplex II type of mechanism that are on the outside of the innermembrane.

Main Sites of Action

Most important main site of action in aerobic cells is the ATP producingelectron transfer system (ETS) in mitochondria. Signals for activatedaerobic metabolism channel from cytosol into nucleus and from there tomitochondria (FIG. 2). Thereafter or simultaneously are MA- andGP-shuttles that shuttle reducing equivalents into the cytosolactivated. Next, the mitochondrial activation of ATP production enhancesthe interplay between mitochondria and endoplasmic reticulum andperoxisomes. Finally increased ATP and substrates (pyruvate and aminogroups) activate inter alia protein and enzyme syntheses; additionallyincreased availability of ATP activates unfolded protein response andsimilar important cellular metabolic control mechanisms.

All in all it seems clearly that the whole ATP energy producing systemof the cell is increased as well as Nrf2/ARE pathways (Example1.1/decline in ROS and Example 1.2.1/increase in NADH concentrations).Rate of beta oxidation seems to be increased also structurally in thelonger term administration (Example 5). Typically beta oxidation andglycolysis compete, but paradoxically also glycolysis may be enhanceddue to increased amount of redox-balancing pyruvate and increasedcapacity of GP- and MA-shuttles.

Produced extra ATP must be consumed, because stored fat or glycogenamounts do not seem to pile up, in fact the contrary (see Example 4).More discussion on these observed effects is in the next section below.

ATP Production Per Gram of Nutrition and Change in Body Composition

ATP is continuously recycled in organisms. It has been estimated thatthe human body, which on average contains some 250 grams of ATP, roughlyturns over its own body weight equivalent in ATP each day.

Even though we can say that aerobic and also anaerobic ATP productioncapacity and very likely also the ATP production is increased by the useof DGA, we can't estimate the precise effect of the use of DGA on theATP production per gram of nutrition because there are so manyendogenous processes that are affected and that feed back to the“equation” that changes also itself.

On one hand GP-shuttles on the outside of inner mitochondrial membrane,can yield increase in metabolic flux with stable ATP production comparedto situation without increased activity of GP-shuttles, i.e. ATPproduction per gram of nutrition is decreased. But on the other handincrease in aerobic energy metabolism within the cells and clearly lesslactate trafficking Example 2.3.3) back to the liver, yields verysignificantly more energy per gram of nutrition compared to glycolysisand lactate cycling/export to the liver. Additionally gene expressionresults (Example 2.3.2) and increased substrate providing to bothshuttles point to that the normal balance between more efficientMA-shuttles and less efficient GP-shuttles is restored to normal verysoon after first spark in GP activity. (Additional note: GP-shuttles arealso limited by its substrate (G-3-P and DHAP) availability in the IMS,and also by their tissue specificity. GP-shuttles may importantly speedup cytosolic NADH oxidation significantly in some situations and in sometissues, but their effect on over all energy metabolism of the body innormal metabolic situations is relatively small.)

Increase in the PPP (G6PD gene) can, depending on the destiny of G6Pmolecule, either increase ATP production (destiny glycolysis) ordecrease it (destiny nucleobase formation). Increase in mitochondrialbeta oxidation of fatty acids by the use of DGA can either enhance ordeteriorate ATP yield per nutrition depending on e.g. how thetriglycerides and related fatty acids are formed. Fatty acid synthesisconsumes NADPH and glyceroneogenesis ATP and NADH. In general betaoxidation is very efficient slow energy provider and thus in normalmetabolic situations its activation increases ATP yield per gram ofnutrition.

All in all it is very reasonable to assume that in vivo, ATP productionpotential per gram of nutrition is increased by some percentage points.Clear reduction in lactate cycling supports this view also.

In example 4 we have seen a surprising phenomenon in which nutritionintake increased statistically significantly in 3 week experiment withrats and simultaneously body weight of rats in DGA groups decreased(males) or remained stable (females) compared to the control groupwithout DGA. The phenomenon comes even more surprising when we assume anincrease in ATP per gram of nutrition. If there is no additional demandfor ATP, it is not produced by the cells. In abundant ATP situations thebody starts to convert nutrition into glycogen and fat (typically astriglycerides) that should increase (not decrease) the weight of therats.

Excess ATP can also be consumed to increased protein synthesis, and tosharpened control of various metabolic processes (e.g. unfolded proteinresponse). As presented FIG. 2 and shown by human (and animal) in vitroand in vivo examples the use of DGA clearly increases the “discussion”between nucleus and mitochondria in energy metabolic andantioxidant/anti-inflammatory pathways. The use of DGA also increasesmitochondrial metabolism and also biogenesis of new mitochondria (andnaturally the autophagy of the old ones). Mitochondria on the other handhave a close interplay between the ER where proteins (rough ER) andlipids (smooth ER) are synthesized. Also peroxisomes are activated bythe use of DGA (see FIG. 1b ). All in all we find it very reasonable tobelieve that the extra (unexplained) ATP is consumed to many anabolicreactions that e.g. in net terms convert fat to protein (muscles) andtriglycerides stores in myocytes (muscles), i.e. fat into muscle mass,and to increased metabolic control leading to clearly healthier over allmetabolism in the long run.

Rough calculation on muscle mass increase. Based on Examples 2.1-2.3(declines in urea cycle and NO output in healthy volunteers) we canassume that the rate of urea output from the body decreases in short(max. 1-4 week) administration of DGA by some 10%. Average men removeroughly 15 grams of urea from the body in the urine per one day. In 15 gof urea there is roughly 7 g of nitrogen (N). In the literature it ismentioned that a gram of N is roughly equal of 6.25 g of protein, and 1g of protein equals roughly 5 g of muscle mass. All in all a 10 per centsave of N due to decline in urea output can be estimated to equal some219 grams of saved (skeletal) muscle mass per day. In adult men skeletalmuscles make up some 42% of the bwt, i.e. some 30 kg in average 70 kgadult man. Thus in 2 weeks over 3 kg or more than 1% of the muscle masscan be saved. This figure can be very likely increased by optimization.In animals DGAcs should probably be used only some weeks or days beforethe slaughtering, because longer term addition on DGAcs into the animalfeed can increase costs uneconomically.

As a non-limiting example, in some optimized conditions meat contents(mass or weight) could possibly increase some 1-2% and simultaneouslyfat contents to drop 2-3% measured of the total body weight with stablefood consumption. Commercially this is viable because the productionprocess by fermentation gives as the side output big amounts of materialthat contains small amounts of DGA and that can be used as food aftervalidation of the production process. Use of the side output increasesalso the environmental sustainability of the production. Additionallythere are potentially even significant possibilities to further enhancethe conversion from fat to protein by the use of DGA by optimization fordifferent life stock, feed, feeding time and DGAcs concentration etc. Anexample being activated interplay between DGA-HPA -loop (genes DGDH andGRHR) and PPP (G6PD) that can in certain settings clearly increasefurther the ATP yield per gram of nutrition.

The Effective Dose and Suitable Time of the Administration

The present inventors have observed in vitro cell culture experimentsand in vivo experiments that administration of DGA, and in vitroexperiment with LGA and HPA (FIG. 11) that in small doses substances ofthe innovation are able to enhance direct and/or indirect mitochondrialmetabolism and simultaneously to reduce oxidative stress. Doses both invivo and in vitro experiments have been adjusted as equal as possible.In here it should be noted that in vitro studies there is full anddirect effect of the substance towards chosen tissues and cells e.g.hepatocytes. In oral administration i.e. in vivo studies used substancefirst enters mouth and gastrointestinal tract and only thereafter to theblood stream etc. Thus most effective “equal” amounts can be severaltimes higher in oral administration compared to cell cultures.

In in vivo studies the doses range from 3 to 12 mg/kg/day, and in invitro studies the doses range from 0.2 to 20 mg/kg/day. Positive effectsfrom the administration are observed with all tested doses (except fortested very low doses in vitro, 0.02 mg/kg/day, in some studies). Aneffective dose depends greatly on the activity of subject'smitochondria. In general the better the physical condition of the userthe smaller doses per kilogram per day are effective. The excess storageof fats and glycogen in the cells may increase the need of the dose perkilogram per day. Based on the results it can be concluded that there isclearly a safe possibility to take bigger doses D-glyceric acidtemporarily, if needed. At the same time it is likely that theincremental positive effects disappear when dose get clearly bigger thannormal effective dose.

L-glyceric acid (LGA) is not a naturally active enantiomer of glycericacid but it can in some circumstances be oxidized into HPA in humans andthus further into DGA. Thus also LGA molecules or its salts and esterscan possess (indirectly) similar positive effects on cell metabolismthan DGA and thus also LGA can be important part of this innovation(FIG. 11, lower graphs).

In vitro experiments with primary human hepatocytes show effects in theamount of ROS, viability and metabolic flux (covering both anabolic andcatabolic reactions). The amount of ROSs in metabolic stress situationis decreased by 15-40% and the viability of cells is increased onaverage by 5-10%. In some cases the hepatocytes viability in vitro hasincreased even by 40-60% (FIG. 10/lower graph). This is a clearindication that DGA has significant impact on the activity of thehepatocytes. That especially metabolic flux, i.e. anabolic and catabolicreactions, is increased is supported by the fact that hepatocytes keptunder starvation (no addition of food, i.e. change of medium during48+1.5-2 h) died clearly more likely than same hepatocytes withoutactivating doses of DGA (first two graphs in FIG. 10).

Several in vivo effects are observed with healthy volunteers afterstandard 10-12 h fasting diet, such as clear increase in plasma pyruvateand clear decrease in lactate levels. Also NO levels were loweredstatistically significantly in healthy volunteers, and increased withone subject experiencing mild to moderate metabolic syndrome (BMI >>25).

Also some enhancement in the intake of glucose, sodium and othernutritional substances from blood to cells is seen. Increased glucoseintake was later confirmed in acute 4.5 day administering in vivo(Example 2.3.4). Significant lowering of bilirubin and bilirubinconjugate in blood is an indication of lower oxidative stress.Importantly in similar test with acute 4.5 day administering of DGA only2.5 hours before the blood sample was taken, also very different resultson bilirubin and HO-1 gene expression were received. With the suitableuse of the DGA it is possible to manage average HO-1 expression up anddown in circadian cycle. Decreased levels of uric acid (UA) in bloodindicate that the systemic oxidative stress of cardiovascular system isreduced (see table 3).

The present invention is based on natural enhancement of indirect anddirect mitochondrial metabolism and mitochondrial energy production. Theamount of reactive oxygen species (ROS) is decreased due to activatedNrf2/ARE systems. The redox state is improved. Also metabolic syndromein general is ameliorated due to increased metabolic flux with less ROS.The positive effect from the use of DGA is obtained during fed andfasting. Fasting situation before going to bed is likely the mosteffective time to promote longer term health effects.

DGA gives cells a signal for increased mitochondrial aerobic metabolism.Simultaneous increase in the activity of mitochondrial NAD⁺ transportingshuttles due to increase in substrates extensifies the positive effecton energy metabolism. Very likely DGA-HPA-loop presented in FIG. 1blengthens the effect of very small administering to last e.g. 24 hbefore a new very small dose is taken. In the longer run also structuralpositive changes towards aerobic metabolism and enhanced capacity forkeeping optimal cytosolic NAD⁺/NADH-ratio will appear, as well asenhanced Nrf2/ARE pathways.

For some individuals, with less active mitochondria, 4 week or evenlonger administration period might be needed for obtaining significantresults like on table 3 for lean persons with good physical condition in4 days. For overweight people or persons with very low physicalcondition the most beneficial combination is to start suitable physicaltraining at the same with DGA administration. This way the healtheffects of DGA arising mostly from mitochondrial activity materializemore rapidly. Additionally daily doses for overweight people or personswith very low physical condition during the first week or two should behigh (7-10 mg/kg twice a day) compared to longer term administration of5 mg/kg once a day before going to bed.

The endogenous antioxidant defense is increased in the body. Thecomposition of the present invention is useful as e.g. a complement tovitamins.

The decrease in ROS and, if needed, the activation of HO-1 geneexpression in cardiovascular system, and simultaneous reduction in bloodpressure, and of blood lactate, sodium, and to some extend also bloodcholesterol may be observed. This leads to improvements in patientssuffering from a cardiovascular disease.

Also patients suffering from cancer and metabolic syndrome may benefitfrom the use of the compositions of the present invention. Anotherexample of a preferable application is use in weight reduction, and inchange of body composition from fat to muscle tissues.

A very useful application of the present invention is in aging processin fighting against neuro- and other degeneration in extremely widerange of diseases and disorders. This is supported by the human in vivoresults and also by in vitro cell studies, wherein genes for increasedenergy metabolism and increased antioxidant defenses andanti-inflammatory response were activated, pyruvate amount was increasedand lactate amount decreased. Also the reduction on the amount of ROS inhuman primary hepatocytes and increase in viability and in some casesincrease of apoptosis of cells were observed supports the idea that theuse of DGA can alleviate, postpone and even heal wide range of agingrelated diseases and/or disorders.

Therapeutic and/or Preventive Pathway(s) Induced by the use of DGA

Enhanced mitochondrial energy metabolism is important for specific andalso pleiotropic effects of the use of DGA or HPA in preventingnon-communicable diseases. Simultaneous daily activation of Nrf2/AREpathway and clear increase in blood pyruvate concentration makes thetherapeutic potential of the use of DGA really significant for extremelywide range of diseases. More than 30% decrease in blood lactate confirmsthe increase in oxidative capacity by the use of DGA. Increase in ATPproduction enhances endogenous cell cycle control and also unfoldedprotein response as well as control of many other metabolic pathways.Extremely important in the use of DGA is its ability to up and also downregulate e.g. HO-1 (Nrf2/ARE) expression during circadian cycle that isclearly seen in conducted two clinical trials (Examples 2.1 and 2.3)with different time of measurements in respect to last DGAcs dosing. Theuse of DGA also promotes homeostasis of protease/antiprotease balance byactivating Nrf2/PERK/MAPK (see FIG. 2).

Cardiovascular Disease

General therapeutic effects for reducing the risk for cardiovasculardiseases: 1) Reducing Oxidative Stress and Inflammation when needed(daily activation of Nrf2/ARE genes). 2) Increasing mitochondrialbiogenesis and energy production of peripheral leukocytes (PGC-1a andNRF1/MT-CO1. also Nrf2). 3) Increasing mitochondrial biogenesis andenergy production of Cardiac Myocytes (PGC-1a and NRF1/MT-CO1, alsoNrf2). 4) Enhancement of liver and kidney function (Nrf2/ARE, decreasedblood urea, decreased blood lactate, improved AST/ALT−, HDL/LDL− andother ratios in blood). 5) Enhancement of lung function againstoxidative stress, activation of Nrf2/ARE. 6) Increasing the viability oferythrocytes, their redox and energy state leading e.g. to increase in2,3-BPG content (Nrf2/ARE and DGA-HPA-loop in FIGS. 2 and 1 b and 3 a).7) Increasing endothelial Nitric Oxide production in subjects in need(Example 2.3.3).

Atherosclerosis

Atherosclerosis is caused by a combination slowly advancing and longlasting events that eventually lead to hardening and narrowing of thearteries due to plaque formation. These causes reduce the elasticity ofthe artery walls but do not affect blood flow for decades because theartery muscular wall enlarges at the locations of plaque. Initial causemay be some tiny defect on artery wall that leads to attack by whiteblood cells to correct the problem. Somehow inflammation is not properlycorrected due to e.g. oxidative stress and/or improper fine tuning ofthe defense response by the body. Deficiency of nitric oxide (NO) andits endothelial synthase (eNOS) enzyme can further escalate the risk ofserious consequences like e.g. elevated blood pressure and eventuallymyocardial infarct (MI). Also excessive amount of LDL compared to HDLcholesterol, and triglycerides in the blood stream may increase the riskof developing atherosclerosis, but there are experts who say that bloodcholesterol and triglycerides can have even contrary effect towardscardiovascular diseases.

It is commonly agreed that oxidative stress and chronic inflammation incardiovascular system are behind slow advance of Atherosclerosis. Alsolack of physical exercise is often one major reason of the advancing ofthis disease. Furthermore consensus exists that nitric oxide (NO) andits endothelial synthase (eNOS) enzyme in endothelial cells and also inRBCs that is a recent discovery can alleviate Atherosclerosis and evenprevent its serious consequences by making artery wall more flexible.

Therapeutic strategy of the use of DGA is to enhance the control ofoxidative stress and reduce it when needed in cardiovascular system bydaily activation of HO-1 and other Nrf2/ARE related antioxidant enzymes.Secondly the use of DGA aims at elevated efficiency in control of theinflammation response by the peripheral leukocytes. This task isachieved by increasing also energy production by increasingmitochondrial aerobic metabolism. Thirdly blood cholesterol balance iskept at suitable range by LDL receptor (LDLR) activation by HO-1expression. Therapeutic effect of the use of DGA is clearly seen inExamples 2.3 and 1.1.2 showing clear increase in PGC-1a/NRF1 relatedgenes, and also in Nrf2/ARE pathway genes (HO-1, G6PD and AOX1).

Prevention of Atherosclerosis follows also from the above describedgeneral therapeutic or preventive pathways. Additionally PGC-1alpha is akey regulator of high glucose-induced proliferation and migration invascular smooth muscle cells (VSMC5), and suggests that elevation ofPGC-1alpha in VSMC could be a useful strategy in preventing thedevelopment of diabetic atherosclerosis.

Positive therapeutic effects from increased physical exercises have beenshown to decrease the risk of Atherosclerosis. The use of DGA providesstrongly similar effects than physical exercise, and thus can preventAtherosclerosis, especially combined with some exercise and normalhealthy diet.

Myocardial Infarction (MI)

MI prevention by the use of DGA follows from prevention ofAtherosclerosis and the above described general therapeutic orpreventive pathways for cardiovascular diseases. As seen in Example 2.1and 2.3 blood levels of Creatine kinase (CK) are down, which is a signof reduced muscle and myocardial dysfunction. Clinical in vivo downregulation of Ck in blood compared to controls also indicates anincreased aerobic ATP production in line with main idea of the use ofDGA. Positive therapeutic effects from increased physical exercises havebeen shown to decrease the risk of myocardial infarction. The use of DGAprovides strongly similar effects than physical exercise, and thus canprevent Myocardial infarction, especially combined with some exerciseand normal healthy diet.

Cardiomyopathy/Congestive Heart Failure

Prevention of cardiomyopathy and congestive heart failure follows fromthe above described general therapeutic or preventive pathways. Positivetherapeutic effects from increased physical exercises have been shown todecrease the risk of cardiomyopathy/congestive heart failure. The use ofDGA provides strongly similar effects than physical exercise, and thuscan prevent cardiomyopathy/congestive heart failure, especially combinedwith some exercise and normal healthy diet.

Vascular Thrombosis and/or Embolism

Over expression of HO-1 has been shown to prevent vascular thrombosisand/or embolism. See also other relevant general stimulation effects bythe use of DGA from above and below.

Asthma and Chronic Obstructive Pulmonary Disease

Chronic Obstructive Pulmonary Disease (COPD) is a term used to describea number of lung conditions that are long-term, gradually worsen, andcause shortness of breath by reducing the normal flow of air through theairways. The most common are emphysema, chronic bronchitis and chronicasthma. Each of these conditions can occur on its own, although manypeople have a combination of conditions. Asthma is a common chronicinflammatory disease of the airways characterized by variable andrecurring symptoms.

Preventive and/or alleviating therapeutic strategy against COPD andAsthma arises from: 1) Enhanced control of oxidative stress in lungs andrespiratory system by daily activation of HO-1 and other Nrf2/ARErelated antioxidant enzymes. 2) Elevated efficiency in control of theinflammation response by respiratory tissues. This task is achieved byNrf2/ARE activation and simultaneous enhancement of energy producingmetabolism by increasing mitochondrial aerobic activity. 3) Increasedsubstrate supply (pyruvate, serine and glycine, see FIG. 1b ) forprotein synthesis, and its enhanced quality and quality control(Nrf2/PERK/MAPK, see FIG. 2). 4) Increase in pyruvate (see pyruvatetherapy above). 5) Increased regeneration of ascorbate (vitamin C) fromdehydroascorbate (DHA), see FIG. 4.

Nrf2 activation can protect lungs from induced acute respiratorydistress syndrome, hyperoxic injury, and in some pulmonary fibrosis byincreasing detoxification pathways and antioxidant defense potential.The use of DGA can increase plasma pyruvate levels by 25% (shown inExample 2.3.3), An increase in plasma is a direct reflection of similarintracellular pyruvate increase through MCTs. increase in pyruvate canalleviate, prevent or even heal many diseases and/or disorders such aslethal sepsis, lethal hemorrhagic shock, Leigh syndrome, COPD and otherinflammatory diseases, mitochondrial DNA depletion and othermitochondrial diseases. The use of DGA can likely efficiently substituteso called pyruvate therapy.

G6PD and 6PGD (6-Phosphogluconate Dehydrogenase) Deficiencies inRBC/hemolytic Anemia and Infant Jaundice,

Severe Nrf2/ARE deficiency has been reported to cause inter aliahemolytic anemia. The use of DGA can alleviate or prevent it activatingNrf2/ARE and enhancement of glycolysis in RBCs. Additionally theactivation of Pentose Phosphate Pathway by the use of DGA may postponeor alleviate G6PD and 6PGD deficiency. Activating DGA-HPA loop in RBCcan also compensate for G6PD and 6PGD deficiency.

Lethal Sepsis and Lethal Hemorrhagic Shock

The use of DGA can increase plasma pyruvate levels by 25% (shown inExample 2.3.3). An increase in plasma is a direct reflection of similarintracellular pyruvate increase through MCTs. Increase in pyruvate canalleviate, prevent or even heal many diseases and/or disorders such aslethal sepsis and lethal hemorrhagic shock.

Elevated Blood Pressure/Hypertension

Primary (essential) hypertension and/or secondary hypertension,including but not limited to incidental hypertension and hypoxicpulmonary hypertension. The use of DGA is an Nrf/ARE pathway/HO-1agonist. Also increased diuresis and natriuresis by the use of DGAreduce hypertension. Other pleiotropic effects from other claims relatedto cardiovascular diseases and also enhanced function of major organs(see above and below) tend to lower elevated blood pressure. Decreasedplasma lactate has been associated at least with reduction of incidentalhypertension. Plasma Nitric Oxide seems to increase in subjects in need.The effect of the use of DGA on elevated blood pressure is seen fromExample 2.1 and FIG. 12.

Hypoxic Pulmonary Hypertension

The alleviating strategy of the invention is based on the above andespecially to the ability of the use of DGA as an efficient HO-1regulator and an efficient agonist with bigger therapeutic doses. Theuse of DGAcs seems to enhance eNOS regulation and thus increase NOlevels in subjects in need.

Disease or Disorder Related to Metabolic Syndrome (if not MentionedElsewhere)

Enhanced mitochondrial energy metabolism is important for specific andalso pleiotropic effects of the use of the DGA also in other diseasesrelated to metabolic syndrome than above mentioned cardiovasculardiseases and hypertension. Simultaneous activation of Nrf2/ARE pathwayand clear increase in blood pyruvate concentration and decrease inlactate makes its therapeutic potential really significant for extremelywide range of diseases related to metabolic syndrome.

Diabetes

Therapeutic effect in diabetes: PGC-1a and NRF1/MT-CO1 relatedenhancement of ATP production in cells. ATP enhances insulin sensitiveGLUT4 cells to facilitate glucose influx (see Example 2.3.4). Glucoseinflux is further assisted by enhanced ATP/energy status of the cellsdue to conversion of extra glucose into glycogen with the help ofphosphate group from UTP (note, ATP+UDP=ADP+UTP). Conversion of glucoseinto glycogen enhances passive diffusion of glucose from plasma. Insulinresistance (IR) is decreased. Significant plasma lactate decrease by theuse of DGA points also clearly towards the ability to postpone or evenheal type II diabetes. Activated AOX1 gene (also belonging to Nrf2/AREpathway) has been shown to detoxify tissues. AOX1 is expressed also inhuman skin, the biggest organ of the body. The lack of AOX1 and ROSscavenging Nrf2/ARE enzymes have been shown to increase IR. Additionaltherapeutic effect of the use of DGA on IR is to activate AOX1 and otherNrf2/ARE enzymes. Pleiotropic effects (1, 3) of the invention towardsall major organs from activation of Nrf2/ARE and PGC-1a/NRF1 pathways,and increase in energy fuel (pyruvate and decrease in lactate) in bloodstream assist prevention of especially type II diabetes. Positivetherapeutic effects from increased physical exercises have been shown todecrease the risk of type II Diabetes. The use of DGA provides stronglysimilar effects than physical exercise, and thus can prevent Diabetes,especially combined with some exercise and normal healthy diet.

Diabetic Neuropathy

Prevention by the use of DGA follows from effects described in sectionDiabetes (above) and from relevant effects described in sectionNeurodegenerative disorders (below).

Disorder Associated with Metabolism

Enhanced mitochondrial energy metabolism is important for specific andalso pleiotropic effects of the use of the DGA, but simultaneousactivation of Nrf2/ARE pathway, and clear decrease of blood lactate andan increase in blood pyruvate concentration makes its therapeuticpotential really significant for extremely wide range of metabolicdiseases, and probably unique in the mechanism of action. Extremelyimportant in the use of DGA is its ability also to down regulate e.g.HO-1 (Nrf2/ARE) expression during circadian cycle like is clearly seenin two different clinical trials and in time dependence compared todosing. Nrf2 serves as a master regulator of the ARE-driven cellulardefense system against oxidative stress. Numerous studies have shownthat Nrf2 protects many cell types and organ systems from a broadspectrum of toxic insults and disease, pathogenesis. Multi-organprotection phenomenon of Nrf2/ARE arises from protection of manydifferent cell types by coordinately up-regulating classic: ARE-drivengenes as well as cell type-specific target genes that are required forthe defense system of each cell type in its unique environment, Thewidespread nature of Nrf2 may have an important therapeutic potential,allowing prevention of also carcinogenesis and neurodegenerativediseases,

Mitochondrial DNA Depletion Syndrome

The use of DGA can increase plasma pyruvate levels by 25% (shown inExample 2.3.3). An increase in plasma is a direct reflection of similarintracellular pyruvate increase through MCTs. It is generally known thatan increase in plasma pyruvate can alleviate, prevent or even heal Leighsyndrome. The use of DGA can likely efficiently substitute so calledpyruvate therapy (see above). Mitochondrial DNA depletion syndrome islikely efficiently alleviated by the direct activation of mitochondrialenergy metabolism and ETS gene RNA expression. Also the activation ofpentose phosphate cycle (G6PD gene expression) and protein synthesisboth support the notion that the use of DGA is likely efficient againstMitochondrial DNA depletion syndrome. (in vivo gene expressions seeExample 2.3.3 and increased protein/enzyme synthesis Examples 2.1-2.3)

Leigh Syndrome

The use of DGA can increase plasma pyruvate levels by 25% (shown inExample 2.3.3). An increase in plasma is a direct reflection of similarintracellular pyruvate increase through MCTs. Increase in pyruvate canalleviate, prevent or even heal many diseases and/or disorders such asLeigh syndrome. Leigh syndrome is related to dysfunction in CNSmitochondria and thus the use of DGA can likely alleviate, prevent oreven heal it also directly by activating mitochondria in the neuronalsystem (Example 2.3.2 and Example 3.2).

Epilepsy

In literature Epilepsy is described as a group of long-term neurologicaldisorders characterized by epileptic seizures. These seizures areepisodes that can vary from brief and nearly undetectable to longperiods of vigorous shaking. In epilepsy, seizures tend to recur, andhave no immediate underlying cause while seizures that occur due to aspecific cause are not deemed to represent epilepsy. Activation ofNrf2/ARE has been shown to alleviate Epilepsy. Also the protection bythe use of DGA against excitotoxic insult could be beneficial forprevention of epileptic seizures. Furthermore inducers of CYP3A4 andCYP2B6 have been used as anticonvulsants and mood stabilizers. InExample 5.3.2 it is shown that the use of DGA can induce both of thesegenes. Preventive and alleviating therapeutic effects of the inventionfor occurrence of epileptic seizures follow from the above andadditionally from general descriptions for age related neurodegenerativediseases (see below).

Bipolar Disorder

Inducers of CYP3A4 and CYP2B6 have been used as anticonvulsants and moodstabilizers in bipolar disorder. In Example 5.3.2 it is shown that theuse of DGA can strongly induce both of these genes, and thus it canpossibly be efficiently used in protection against bipolar disorder andpossibly also in schizophrenia. For additional preventive andalleviating therapeutic effects of the invention for occurrence ofbipolar disorder, see epilepsy (above) and general descriptions for agerelated neurodegenerative diseases (below).

Psychiatric Disorders and Mood Disorders

Psychosis, schizophrenia, autism, depression, personality change, panicdisorder, anxiety disorder. Major psychiatric diseases are common,chronic, recurrent mental disorders that affect the lives of millions ofindividuals worldwide. Although schizophrenia and mood disorders are notclassic neurodegenerative disorders, there is an increasing amount ofevidence that these disorders are associated with abnormalities incellular plasticity, including the ability of neuronal and glial cellsto resist or adapt to environmental stressors and the ability of thesecells to undergo remodeling of synaptic connections. Neuronal functionis highly dependent on mitochondrial function. Thus impairedmitochondrial function might lead to a disruption of normal neuralplasticity and reduce cellular resilience, promote the development orprogression of mood and psychotic disorders. The use of DGA canalleviate or prevent psychiatric and mood disorders by activating itsthree main pathways Nrf2/ARE, PGC-1a/NRF1 and pyruvate formationpresented in FIG. 2.

Cerebrovascular Accident, Damage from Acute Head Injury

Prevention of Cerebrovascular accident (CVA, or alternatively stroke orischemia) follows from the above described general therapeutic orpreventive pathways. Also enhanced recovery and damage suppressionfollows from the above described general therapeutic or preventivepathways. The protection of neurons against excitotoxic insult by theuse of DGA, see example 3.1, is beneficial for prevention of permanentinjuries from CVA and other damage from acute head injury or trauma.

Acute or Chronic Renal Failure

Nrf2/ARE has been shown to increase diuresis and natriuresis (also seenin clinical tests, Example 2.1-2.3) that indicates improvement in renalfunction. Activated AOX1 gene has been shown to detoxify varioustissues. Therapeutic effects of the use of DGA on renal function arisealso from reduced/shared burden on kidneys to detoxify body fluids. E.g.blood urea levels have been shown to decrease after the use of the useof DGA. The use of DGA facilitates also the prevention of diabeticnephropathy in chronic kidney disease. Kidneys have also a role ingluconeogenesis, secondary to the liver. Significantly decreased lactatelevels and increased amount of pyruvate in blood stream by the use ofDGA is a clear indication that the pressure towards gluconeogenesis inthe liver and in kidneys for brains and other tissues decreases. Itliberates renal resources for other important metabolic functions,especially in subjects in need. By rendering free capacity to kidney's,and the activation of Nrf2/ARE and aerobic energy metabolism the use ofDGA suppresses and corrects various renal malfunctions. Thus it maypossibly also reduce e.g. kidney stone formation due to activation ofmetabolic control.

Acute or Chronic Liver Failure

Liver is probably the most important inner organ in metabolism. On topof various vital tasks related to nutrition intake and excretion, andmetabolite detoxifying, it has a major role in gluconeogenesis andtriglyceride synthesis, and numerous other tasks that effect wellbeingof other major organs and whole physiological system(s). Significantlydecreased lactate levels and increased amount of pyruvate in bloodstream by the use of DGA is a clear indication that the pressure towardsgluconeogenesis in the liver decreases. It liberates hepatic resourcesfor other important metabolic functions, especially in subjects in need.

In Examples 1.1-1.2 and 2.1-2.3 we show clearly that the use of DGAactivates very effectively endogenous ROS scavenging mechanisms in humanprimary hepatocytes. In separate gene expression studies withhepatocytes (in vitro) and peripheral leukocytes (in vivo) we have beenable to show that ROS scavenging is due to increased expression ofNrf2/ARE related genes e.g. HO-1, G6PD and AOX1. Also the masterregulator PGC-1a/NRF1 gene was activated in human primary hepatocytes.

Test set up with hepatocytes (in vitro) in Starving Diet and thecomparison with standard diet indicates clearly that hepatic activityincreases by the invention and it leads to cell death due to lack ofnutrition, i.e. starvation in 48 h test. Significantly increased levelsof circulating triglycerides in some of the clinically testedindividuals (Examples 2.1-2.3) indicate also enhanced hepatictriglycerides formation activity by the use of DGA in vivo. (Theincrease in triglycerides is of course also an indication of increaseddemand of fatty acids for beta oxidation, resulting further in increasein hepatic and plasma free glycerol levels (see Example 5).

Splenomegaly

Nrf2/ARE deficiency has been reported to cause Splenomegaly and oftenrelated hemolytic anemia. The use of DGA can alleviate or preventSplenomegaly by activating its three main pathways Nrf2/ARE, PGC-1a/NRF1and pyruvate formation presented in FIG. 2.

Increased viability of RBCs (see e.g. table 4/LDH result and FIG. 3.d)can reduce the risk of Splenomegaly.

Acute or Chronic Pancreatic Failure

Increase in PGC-1a/NRF1 pathway and subsequent enhancement in energymetabolism alleviates symptoms. Interestingly it has been shown thatGP-shuttles are especially active in pancreatic beta cells that produceinsulin. GP-shuttles and increase in aerobic metabolism produce ROS. Onthe other hand Nrf2/ARE -mediated antioxidant induction has been shownto play paradoxical roles in pancreatic beta-cell function: 1) inductionof antioxidant enzymes protects beta-cells from oxidative damage andpossible cell death, thus minimizing oxidative damage related impairmentof insulin secretion, and 2) the induction of antioxidant enzymes byNrf2 activation blunts glucose-triggered ROS signaling, thus resultingin reduced glucose stimulated insulin secretion.

In our limited clinical tests blood insulin levels have tended toincrease more rapidly with the use of the invention, thus indicatingactivated pancreatic function.

Chronic Auto Inflammation and Autoimmune Syndrome and Diseases

Initial cause of the onset of chronic auto inflammation and autoimmunediseases may be some tiny defect somewhere in the body that leads toattack by the immune defense system to correct the problem. Somehowinflammation is not properly corrected due to e.g. oxidative stressand/or improper fine tuning of the defense response by the body. Alsosome long term irritation from external toxins can cause the onset ofthe syndrome.

In literature Auto inflammatory diseases are described as a relativelynew category of diseases that are different from autoimmune diseases.However, autoimmune and auto inflammatory diseases share commoncharacteristics in that both groups of disorders result from the immunesystem attacking the body's own tissues, and also result in increasedinflammation.

Therapeutic strategy of the use of DGA is to enhance the oxidativestress and the anti-inflammation control of tissues and also the immunesystem by daily ensuring and testing of the activity of Nrf2/ARE pathwayenzymes. Secondly the use of DGA aims at elevated efficiency in controlof the inflammation response by increasing aerobic energy production ofperipheral leukocytes and immune systems as a whole. This task isachieved by increasing mitochondrial aerobic metabolism, i.e.PGC-1a/NRF1 related genes. Therapeutic effect of the use of DGA isclearly seen in examples 2.3.3 (and 1.2) showing clear increase inPGC-1a/NRF1 related genes, and also in Nrf2/ARE pathway genes (HO-1,G6PD and AOX1). The use of DGA increases also detoxifying capacity byincreasing AOX1 gene expression.

Furthermore, the use of DGA can decrease NO levels in the bodydemonstrated in example 2.3.3. In literature it has been reported thatiNOS induced NO (see description of FIG. 4) often exacerbatesinflammation, thus the reductions can be beneficial. The statisticallysignificant 13% decline in NO in healthy individuals is likely due toinhibition of NF-kB (FIG. 2) that can explain the reduced iNOS activityand subsequent reduction in NO by the use of DGA.

Finally the use of DGA can increase plasma pyruvate levels by 25% (shownin example 2.3.3). An increase in plasma is a direct reflection ofsimilar intracellular pyruvate increase through MCTs. Increase inpyruvate can alleviate, prevent or even heal many inflammatory diseases.Non-limited examples of chronic auto inflammation diseases: 1) Gout is amedical condition usually characterized by recurrent attacks of acuteinflammatory arthritis—a red, tender, hot, swollen joint. 2) Rheumatoidarthritis is an autoimmune disease that results in a chronic, systemicinflammatory disorder that may affect many tissues and organs, butprincipally attacks flexible (synovial) joints.

Reduction in uric acid may alleviate gout as well as increase in aerobicenergy metabolism. Also the ability of DGA to manage HO-1/Nrf2 enzymesup and down paves way for efficient therapy regimen towards arthritisgout and also rheumatoid arthritis. See also other general therapeuticeffects of the use of DGA (in above and below) for alleviating andprevention of Chronic auto inflammation and autoimmune syndrome anddiseases.

In this use the DGA can be classified as NSAID, i.e. non-steroidalanti-inflammatory drug.

Psoriasis

Psoriasis is an inflammatory skin disease with characteristic changes inthe epidermis that resembles unsuppressed wound healing due to excessivehyperproliferation of keratinocytes. The exact cause of PsoriasisVulgaris and Psoriasis Arthritis is not known but dysfunction in cellregulation due to ROS and toxins, and possibly due to dysfunction inenergy metabolism are important reasons for onset and incidence ofpsoriasis. In recent years there has arisen growing evidence thatpsoriasis more frequent in patients with other disorders related e.g. tometabolic syndrome.

Therapeutic effect 1a: Increase in the activity of HO-1/Nrf2/ARE relatedenzyme pathway against epithelial ROS damage.

Therapeutic effect 1b: In literature it has been shown that increasedHO-1 expression is essential for wound healing indicating clearly thatthe use of DGA has the potential to manage occurrence of Psoriasis dueto the fact that it can efficiently and dose dependently manage HO-1expression in cells, example 2.1, 2.3.1 and 2.3.2.

Therapeutic effect 2: Increase in the activity of AOX1/Nrf2/ARE relatedenzyme pathway against various toxins towards epithelial cells.

Therapeutic effect 3: More accurate control and suppression of overexpression of auto inflammatory pathways and cell proliferation due toenhanced energy production (pyruvate therapy) and status of epithelialkeratinocytes.

Therapeutic effect 4: the use of DGA can decrease NO levels in the bodydemonstrated in example 2.3.3. It is well known that iNOS induced NO(see description of FIG. 4) often exacerbates inflammation and clearlyobserved also in Psoriasis, thus observed reductions in NO can bebeneficial.

Therapeutic effect 5: Pleiotropic effect from management of generaldisorders related e.g. to Metabolic Syndrome can likely postpone theonset or reduce the occurrence of Psoriasis.

Impairment in Collagen Synthesis

Collagen is the most abundant protein in mammals. Its synthesis istypically reduced as an organism ages. From the literature any defect incollagen synthesis is described to lead to disorders like impairedosteogenesis, scurvy, systemic lupus erythematosus as well as some otherauto immune diseases. Reduced synthesis of collagen types I and III isalso characteristic of chronologically aged skin.

Many cell types e.g. osteoblasts produce collagen. Glycine is clearlythe most abundant amino acid in collagen. It forms approximately onethird of its content. Reduced form of ascorbate is a rate limiting stepin collagen synthesis.

The invention can 1) enhance stem cell differentiation into osteoblastsby increased HO-1 expression (Example 2.3.2), 2) very likely increaseperoxisomal glycine and pyruvate output from imported alanine andglyoxylate via HPA-Serine-loop (see FIG. 1 b, and FIG. 3) canefficiently enhance ascorbate regeneration from dehydroascorbate(ROS/Example 1.1 and increased NADPH producing capacity, FIG. 4).

Thus the use of DGA can attenuate, postpone, and even cure many agerelated diseases arising from impaired collagen synthesis.

Pre-Eclampsia

Pre-eclampsia is a disorder of pregnancy characterized by high bloodpressure and large amounts of protein in the urine. Though present inthe majority of cases, protein in the urine need not be present to makethe diagnosis of preeclampsia. It involves many body systems andevidence of associated organ dysfunction may be used to make thediagnosis when hypertension is present.

From the literature, serum concentration of oxidized low-densitylipoprotein (oxLDL) is higher in women with preeclampsia than in normalpregnant woman. LDL receptor (LDLR) is the scavenger receptor for oxLDLand it is abundantly expressed in placenta. It is well known that oxLDLactivates nuclear factor erythroid 2-related factor 2 (Nrf2), a masterregulator of antioxidant and cytoprotective genes such as hemeoxygenase-1 (HO-1), which play an important role in pre-eclampsia. HO-1activation has been shown to alleviate pre-eclampsia.

With correct dose, the use of DGA can significantly increase HO-1expression in peripheral leukocytes and other tissues. Additionalimportant therapeutic effect from the use of DGA comes from itsalleviating effects towards other organ dysfunction related topre-eclampsia.

Thyroid Disease

In literature Thyroid disease is described as a medical conditionimpairing the function of the thyroid gland. Imbalance in production ofthyroid hormones arises from dysfunction of the thyroid gland itself, orthe pituitary gland, which produces thyroid-stimulating hormone (TSH),or the hypothalamus, which regulates the pituitary gland viathyrotropin-releasing hormone (TRH). Concentrations of TSH increase withage, requiring age-corrected tests. Hypothyroidism affects between threeand ten percent of adults, with incidence higher in women and theelderly.

The use of DGA can alleviate and postpone the onset of dysfunction ofthyroid gland. Therapeutic strategy of the use of DGA is to enhance theoxidative stress and the anti-inflammation control of tissues and alsothe immune system by daily activation of Nrf2/ARE pathway enzymes.Secondly the use of DGA aims at elevated efficiency in control of theinflammation response by increasing aerobic energy production ofperipheral leukocytes and immune systems as a whole. This task isachieved by increasing mitochondrial aerobic metabolism, i.e.PGC-1a/NRF1 related genes. Therapeutic effect of the use of DGA isclearly seen in gene expression and other examples showing clearincrease in PGC-1a/NRF1 related genes, and also in Nrf2/ARE pathwaygenes (HO-1, G6PD and AOX1). The use of DGA increases also detoxifyingcapacity by increasing AOX1 gene expression.

Chronic Fatigue

Main therapeutic effect: increase in energy metabolism and production ofATP (PGC-1a/NRF1 pathway).

Positive therapeutic effects from increased physical exercises have beenshown to decrease the symptoms of chronic fatigue. The use of DGAcsprovides strongly similar effects than physical exercise, and thus canalleviate and even prevent chronic fatigue, especially combined withsome exercise and normal healthy diet.

Fibromyalgia

Main therapeutic effect: increase in energy metabolism and production ofATP (PGC-1a/NRF1 pathway).

Also increased Nrf2/ARE -mediated antioxidant defense andanti-inflammation control alleviate fibromyalgia.

Positive therapeutic effects from increased physical exercises have beenshown to decrease the symptoms of chronic fatigue. The use of DGAcs(Invention) provides strongly similar effects than physical exercise,and thus can alleviate and even prevent chronic fatigue, especiallycombined with some exercise and normal healthy diet.

Overweight

The use of DGAcs increases anabolic and anaplerotic reactions that perse promote healthier metabolism and on the other hand consume a lot ofenergy. Used energy is consumed in the form of ATP (e.g. in pyruvatecarboxylase, protein synthesis), GTP (e.g. in gluconeogenesis), UTP(e.g. glycogen production), and CTP (for phospholipid synthesis).Importantly ATP can render its phosphate group to GDP, UDP and CDP usingenzyme nucleoside-diphosphate kinases and activate them back to theirmost active/highest energy state. Thus enhanced ATP production of theuse of DGA can efficiently contribute to these anabolic and anapleroticreactions. Increased use of energy/nutrition leading to weight loss bythe invention has been shown indirectly in controlled starving diet testwith human primary hepatocytes and calorie restriction test with ratcortical neurons, see. Examples 1.1 and 3.1, and also directly in 3weeks rat feeding study, see Example 4. The use of DGA works best toreduce overweight when also aerobic muscle cells (myocytes) areactivated by exercise, i.e. when the main pathways related to the use ofDGA are in use.

Cancer

Increase in antioxidant protection of basically all cell types fromactivation of Nrf2/ARE, i.e. decreased DNA damage from ROS and thusdecline in occurrence of cancer cells. Enhanced energy metabolism andmitochondrial activity leads to more accurate control of malignantcancer cells and their controlled apoptosis. Also enhanced pyruvatesupply to aerobic cells facilitates cells' endogenous quality control.Nrf2/ARE/HO-1 as an inhibitor of NF-kB nuclear translocation, canincrease apoptosis in certain cancers, e.g. solid tumor andhematological malignancies. On the other hand NF-kB activation can alsohave positive effect for suppressing some tumors. Enhanced aerobicenergy production of potential tumor cells by the use of DGA facilitatescritical screening process for controlled apoptosis.

Cancer Subtypes

Basically all types of cancers that are caused by ROS damage to thecell, dysfunctioning mitochondria (e.g. compromised ability forapoptosis) or/and dysfunction of the energy production of the cells canbe postponed or even prevented by the invention. In some cases the useof DGA could even facilitate a process that endogenously could suppresssome tumor. In general by alleviating aging related degeneration of thecells, the use of DGA can reduce the number of malignant cells and/orenhance their controlled cell death.

List of cancer subtypes that the use of DGA can supposedly postpone,alleviate, prevent or even suppress can be found e.g. in National CancerInstitute of US NIH: www.cancer.gov/cancertopics/types/alphalist

Disease or Disorder Related to Aging of an Organism, i.e. Degenerationof the Organism, its Organs, and Cell Tissues Like Neuronal, Epithelial,Endothelial, and Other Metabolically Active Tissues

General Therapeutic or Preventive Effects and Pathways of the Inventionfor Diseases and Disorders Related to Aging

Enhanced mitochondrial energy metabolism (PGC-1a/NRF1) is important forspecific and also pleiotropic effects of the use of DGA in preventingdegeneration due to aging. Simultaneous activation of Nrf2/ARE pathwaysgenes is also crucial. Additionally simultaneous clear increase in bloodpyruvate concentration makes therapeutic potential of the use of DGAreally significant for extremely wide range of ageing related diseasesand probably unique in the combined action of multiple mechanisms.Related increase on mitochondrial biogenesis in neurons, leukocytes, andother aerobically active cell types intensifies the defensive mechanismsagainst degeneration due to aging of an organism. Extremely important inthe use of DGA is its ability also to down regulate e.g. HO-1 (Nrf2/ARE)expression during circadian cycle. This is clearly seen in conducted twoclinical trials, with different time of measurements compared to dosingof the use of DGA, Examples 2.1 and 2.3.3. Decrease in diabeticneuropathy can prevent slowly advancing chronic neurological diseases.(See above)

Age Related Hearing Loss

Age related hearing loss includes but is not limited to presbyacusis,Nnise induced hearing impairment and ototoxic hearing impairment.Age-related hearing loss (AHL) is associated with an age-dependent lossof sensory hair cells, spiral ganglion neurons, and stria vasculariscells in the inner ear. AHL is thought to be the result of aging,oxidative damage, mitochondrial impairment, and environmental factors.Noise is the most documented environmental factor causing hearing loss.Outer hair cells are the primary lesion from noise exposure, and theaccumulated effect of noise is thought to contribute to AHL. Ototoxicsubstances such as aminoglycoside antibiotics also increasesusceptibility to AHL as these drugs can damage hair cells.

Experimental evidence suggests that mitochondrial dysfunction associatedwith reactive oxygen species (ROS) plays a central role in the agingprocess of cochlear cells. Cochlear cells are exquisitely sensitive todisturbances in energy metabolism. There is a growing bod of evidencesuggesting mitochondrial ROS contributes to AHL that is age-dependentand has no defining genetic basis.

Reactive oxygen species contribute to the formation of several types ofcochlear injuries, including age-related hearing loss and medicineinduced ototoxity. The present findings in literature clearly indicatethat Nrf2/ARE pathway protects the inner ear against age related hearinginjuries and ototoxicity by up-regulating antioxidant enzymes anddetoxifying proteins.

Therapeutic effect 1: Longer term managed increase in the activity ofHO-1/Nrf2/ARE related enzyme pathway against hair cell ROS damage.

Therapeutic effect 2: Increase in the activity of whole Nrf2/ARE pathwayand related up-regulation of detoxifying enzymes in the inner ear.

Therapeutic effect 3: Increase in PGC-1a/NRF1 pathway and subsequentenhancement in energy metabolism could postpone or alleviates symptomsof age-related hearing loss. Enhanced neuronal metabolism (due also toincreased pyruvate) can also facilitate signal transportation from haircells to the brains.

Age Related Macula Degeneration

From the literature: Age related macular degeneration (AMD) is triggeredby oxidative stress, which imbalances innate immunity. Retinal pigmentepithelial (RPE) cell/mitochondrial damage are the key events in earlydisease that is regulated by the transcription factor Nrf2/ARE pathway.Impaired Nrf2 signaling induces mitochondrial and RPE dysfunction thatresults in an oxidative, inflammatory, and pathologic microenvironment.

Preventive therapeutic effect 1: Increasing mitochondrial biogenesis andenergy production in RPEs indicated by similar increase in neurons,peripheral leukocytes and hepatocytes (genes: PGC-1a and NRF1/MT-CO1,GPD2 and also Nrf2/ARE genes).

Preventive therapeutic effect 2: reduction in oxidative stress byincreasing the activity of HO-1 and other Nrf2/ARE dependentantioxidants.

Therapeutic effect 3: Pleiotropic effect from management of generaldisorders related e.g. to Metabolic Syndrome (see above) can likelypostpone the onset of AMD.

Glaucoma, Optic Neuropathy and Ischemic Optic Neuropathy

Glaucoma is a term describing a group of ocular disorders withmulti-factorial etiology united by a clinically characteristicintraocular pressure-associated optic neuropathy. This can permanentlydamage vision in the affected eye(s) and lead to blindness if leftuntreated.

The nerve damage involves loss of retinal ganglion cells in acharacteristic pattern. The many different subtypes of glaucoma can allbe considered to be a type of optic neuropathy.

Preventive therapeutic effect 1: Increasing mitochondrial biogenesis andenergy production in optical neurons, ganglia cells, RPEs etc. indicatedby similar increase in neurons, peripheral leukocytes and hepatocytes,i.e. genes: PGC-1a and NRF1/MT-COO1, GPD2 and also Nrf2/ARE genes, seeExamples 1, 2, and 5)

Preventive therapeutic effect 2: reduction in oxidative stress byincreasing the activity of HO-1 and other Nrf2/ARE dependentantioxidants.

Therapeutic effect 3: Pleiotropic effect from management of hypertensionand diabetes (see above) can likely postpone the onset of Glaucoma andacute Glaucoma, i.e. Ischemic Optic Neuropathy and/or Optic Nerve Crash.For Ischemic optic neuropathy, see also cerebrovascular accident above.

Increase in glycerol by the use of DGA may alleviate acute glaucoma (seeexample 5).

Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a prevalent cause of blindness caused by alarge number of different mutations in many different genes. Themutations result in photoreceptor cell death of the retina. It has beenwidely suggested that oxidative stress possibly contributes to itspathogenesis.

Preventive therapeutic effect 1: reduction in oxidative stress byincreasing the activity Nrf2/ARE dependent antioxidants.

Preventive therapeutic effect 2: Increasing mitochondrial biogenesis andenergy production in rod and cone cells indicated by similar increase inneurons, peripheral leukocytes and hepatocytes (genes: PGC-1a andNRF1/MT-CO1, GPD2 and also Nrf2/ARE genes).

Preventive therapeutic effect 3: Increase in blood pyruvateconcentration increases the viability of rod and cone cells.

Sarcopenia

Sarcopenia is described as the age-associated decline in muscle mass.The physical basis for the disorder is a combination of atrophy, loss ofthe constituent muscle fibers, and defects in energy metabolism inskeletal muscle. Metabolic genes and myosin isoform expression areregulated through the transcriptional co-activator PGC-1a. Changes inmuscle metabolism can have systemic effects. It has been shown thatdefects in skeletal muscle energy metabolism are linked to type IIdiabetes and glucoregulatory dysfunction.

The compositions and methods of the present invention can alleviate orprevent sarcopenia by activating its three main pathways Nrf2/ARE,PGC-1a/NRF1, and pyruvate formation presented in FIG. 2.

Osteoporosis

Osteoporosis is described as a progressive bone disease that ischaracterized by a decrease in bone mass and density which can lead toan increased risk of fracture. In osteoporosis, the bone mineral density(BMD) is reduced, bone microarchitecture deteriorates, and the amountand variety of proteins in bone are altered.

The use of DGA can increase the differentiation in mesenchymal stemcells into osteoblasts (instead of developing into adipocytes) byenhancing overexpression of HO-1 gene and thus attenuate osteoporosis bypromoting bone formation.

Also enhancing de novo synthesis of vitamin D precursors throughfacilitating Mevalonate Pathway by increasing NAPDH supply (see FIG. 3band FIG. 5) may postpone Osteoporosis.

On the other hand the use of DGA also may attenuate osteoporosis byenhancing general metabolism, aerobic energy formation, and related ROSformation by increasing PGC-1a/NRF1 gene expression and daily testing ofNrf2/ARE pathways. Also internal control of excess or in-excess boneformation and turnover may be enhanced by the use of DGA due toincreased aerobic energy supply.

De novo increase in pyruvate and other building blocks for anabolicreactions, and simultaneous increase in required energy (ATP, GTP, UTPand CTP) for anabolic reactions by the use of DGA, likely plays a rolein preventing or postponing the onset of Osteoporosis.

Osteoarthritis

Osteoarthritis (OA) also known as degenerative arthritis or degenerativejoint disease or osteoarthrosis, is a group of mechanical abnormalitiesinvolving degradation of joints, including articular cartilage andsubchondral bone.

Therapeutic strategy of the use of DGA for osteoarthritis is to enhancethe metabolism and renovation of lost tissues, especially collagen. Incollagen synthesis vitamin C, in its active reduced form, is anessential co-factor. The ability of the invention to promoteregeneration of ascorbic acid from e.g. dehydroascorbate possesses thespecific therapeutic effect for preventing and curing osteoarthritis.Additionally the use of DGA aims at elevated efficiency of metabolism byincreasing aerobic energy production and by increasing anabolicreactions for all protein, including collagen, synthesis. This task isachieved by increasing mitochondrial aerobic metabolism, i.e.PGC-1a/NRF1 related genes, and resulting increased amount of pyruvate inthe cells and blood circulation. Therapeutic effect of the use of DGA isclearly seen in Examples 1.2.3 & 2.3.3 showing clear increase inPGC-1a/NRF1 related genes. Also increase in gene expression of G6PD isan indirect sign of increased NADPH production and indirectly also onelevated anabolic reactions due to Acetyl Coa signaling (FIG. 3b .).

Aging Related Neurodegenerative Diseases in General

On top of general effects for age related diseases described above,additional important general therapeutic effect for neurologicaldysfunctions is the increase concentration of pyruvate in blood by theuse of DGA. CNS needs a lot of energy for sustaining adequate neuronalsignaling provided by axons and synapses. Especially axonal activitybenefits from increased external energy source from pyruvate. Pyruvateis transported through blood brain barrier via transport system. Forpreventing age related neuronal diseases Nrf2/ARE expression inastrocytes, and its protective role towards oxidative stress also inneurons is very important. Also positive therapeutic effects fromincreased physical exercises have been shown to prevent, and at least todecrease the symptoms of Neurodegenerative diseases. The use of DGAprovides strongly similar effects than physical exercise, and thus canalleviate and even prevent Neurodegenerative diseases, especiallycombined with some exercise and normal healthy diet.

As a whole the use of DGA is very suitable in prevention, alleviation,and even curing of a wide range of age related neuronal disorders.Indirect evidence from clinical in vivo testing indicates that the useof DGA possesses very clear effect positive towards the CNS in general.In vitro excitotoxity studies with primary rat cortical neurons confirmthat there exists also a protective effect from the use of DGA, as wellas statistically significant increase in mitochondrial biogenesis.

Chronic Neurodegeneration

Chronic neurodegeneration is the umbrella term for the progressive lossof structure or function of neurons, including death of neurons. Manyneurodegenerative diseases including ALS, Parkinson's, Alzheimer's, andHuntington's occur as a result of neurodegenerative processes. Asresearch progresses, many similarities appear that relate these diseasesto one another on a sub-cellular level. Discovering these similaritiesoffers hope for therapeutic advances that could ameliorate many diseasessimultaneously. There are many parallels between differentneurodegenerative disorders including atypical protein assemblies aswell as induced cell death.

Preventive and alleviating therapeutic effects of the invention followfrom above general descriptions for age related neurodegenerativediseases.

Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease withvarious causes. It is characterized by muscle spasticity, rapidlyprogressive weakness due to muscle atrophy, difficulty in speaking(dysarthria), swallowing (dysphagia), and breathing (dyspnea).

Preventive and alleviating therapeutic effects of the invention followfrom above general descriptions for age related neurodegenerativediseases.

Additionally in prevention of ALS especially important is the enhancedaxonal energy metabolism by the use of DGA. The use of DGA supportswellbeing of oligodendrocytes and their important role in axonalintegrity and wellbeing.

Alzheimer's Disease

Alzheimer's disease (AD) is the most common cause of dementia. The termdementia describes a set of symptoms which can include loss of memory,mood changes, and problems with communication and reasoning.Neurodegenerative processes associated with AD are complex and involvemany CNS tissue types, structures and biochemical processes. Factorsbelieved to be involved in these processes are generation of ROS,associated inflammatory responses, and the bio-molecular and geneticdamage they produce. Furthermore beta-amyloid formation in the brainshas been considered one important cause of AD. It has been shown thatincreased activity of PGC-1a can suppress BACE1 expression that is theenzyme behind beta-amyloid formation. Also activation of themitochondrial ETC especially MT-001, has been related to prevention ofAD. The invention can increase PGC-1a and MT-001 activity, see Example5.3.2. Nrf2/ARE pathway activation has been shown to decrease spatiallearning difficulties related to AD in mouse model.

The use of DGA has been shown in vivo or in vitro to specificallyaddress to all above mentioned general causes of AD, and thus itpossesses at least preventive and/or alleviating effect on AD.Additional effect on prevention of excessive beta-amyloid formation isreduced ER stress by the use of DGA (see graph 2). In literature ERstress has been suggested to be involved in some human neuronaldiseases, such as Parkinson's disease, Alzheimer's and prion disease.

Parkinson's Disease

Parkinson's disease (PD) is characterized by the progressive loss ofspecific cells of the brain region called substantia nigra that producethe chemical messenger dopamine. The current mainstay therapy is theadministration of drugs that mimic dopamine action.

The main strategy of the use of DGA is in preventing PD is theadministration of therapies aimed to prevent neuronal cell death. Theuse of DGA has been shown in vitro to protect cortical neurons againstexcitotoxic insults by NMDA stimulation. Also neurons of substantianigra possess NMDA receptors and thus it is reasonable to believe thatthe use of DGA can increase their viability and thus prevent oralleviate PD, and/or even cure early PD.

In literature there are reports that PD might be caused by defect incomplex I of the ETS or some other mitochondrial defect. As has beenshown the use of DGA enhances mitochondrial metabolism (GPD2,MT-CO1/NRF1) and mitochondrial biogenesis specifically in neurons, andthus can possess therapeutic effect for PD also from that angle. Relatedto enhanced energy metabolism also PGC-1a has been implicated as apotential therapy for PD.

Additionally also Nrf2/ARE enhancement of the use of DGA can likely beused as efficient PD therapy.

Reduced ER stress and enhanced energy metabolism of neurons might alsosuppress the formation of Lewy Bodies in pathological conditions of PD.

Multiple Sclerosis

In literature Multiple sclerosis (MS) is described as an inflammatorydisease in which the insulating covers of nerve cells in the brain andspinal cord are damaged. This damage disrupts the ability of parts ofthe nervous system to communicate, resulting in a wide range of signsand symptoms. Preventive and alleviating therapeutic effects of theinvention for MS follow from above general descriptions for age relatedneurodegenerative diseases (e.g. enhancement of Nrf2/ARE pathway).

Especially important in prevention and alleviating the symptoms of MS isthe enhanced axonal energy metabolism by the use of DGA. The use of DGAsupports wellbeing of oligodendrocytes, and other glial cells in theirimportant role in axonal integrity and wellbeing. Thus the use of DGAenhances communication capabilities of the nervous system that is oftenimpaired in MS.

Huntington's Disease

In the literature it is shown that Huntington's disease (HD) is causedby an expansion of cytosine-adenine-guanine (CAG) repeats in thehuntingtin gene, which leads to neuronal loss in the striatum and cortexand to the appearance of neuronal intranuclear inclusions of mutanthuntingtin. Huntingtin plays a role in protein trafficking, vesicletransport, postsynaptic signaling, transcriptional regulation, andapoptosis. Thus, a loss of function of the normal protein and a toxicgain of function of the mutant huntingtin contribute to the disruptionof multiple intracellular pathways. Furthermore, excitotoxicity,dopamine toxicity, metabolic impairment, mitochondrial dysfunction,oxidative stress, apoptosis, and autophagy have been implicated in theprogressive degeneration observed in HD.

Preventive therapeutic effect: Increased expression of PGC-1a andmitochondrial metabolism, combined with reduced ROS generation by theuse of DGA work in the direction of postponing the age of onset of HD.

Prion Disease

Prion disease represents a group of conditions that affect the nervoussystem. These conditions impair brain function, causing changes inmemory, personality, and behavior; a decline in intellectual function(dementia); and abnormal movements, particularly difficulty withcoordinating movements (ataxia).

Preventive and alleviating therapeutic effects of the invention forPrion diseases follow from above general descriptions for age relatedneurodegenerative diseases.

In literature specifically ER stress has been suggested to be involvedin some human neuronal diseases, such as Prion disease. The use of DGAcan reduce ER stress (see FIG. 2) and thus likely alleviate itssyndromes or even postpone the onset of this rare disease.

In aspect 1 the invention provides a composition comprising one or morecompounds selected from the group consisting of D-glyceric acid,DL-glyceric acid, L-glyceric acid, and hydroxypyruvatic acid and saltsand esters thereof for use in a method of treating or preventing anon-communicable disease or disorder related directly or indirectly tomitochondrial degeneration and/or mitochondrial dysfunction, impairedcytosolic catabolism of carbohydrates, deteriorated antioxidantdefenses, deteriorated inflammation control, formation of malfunctioningproteins, and/or decreased ability to synthesize precursors ofnucleobases adenine and/or guanine.

Aspect 2 provides a composition comprising one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, and hydroxypyruvatic acid and salts and esters thereoffor use in a method of treating or preventing a disease or disorderaccording to aspect 1, wherein the disease or disorder is acardiovascular disease, metabolic syndrome, disorder associated withmetabolism, cancer, overweight, elevated blood pressure, or adegeneration disease related to the aging process of an organism, or adegeneration disease accelerating the aging process of an organism.

Aspect 3 provides a composition comprising one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, and hydroxypyruvatic acid and salts and esters thereoffor use in a method of treating or preventing a disease or disorderaccording to aspect 2, wherein the cardiovascular disease is selectedfrom the group consisting of atherosclerosis, myocardial infarction,cardiomyopathy or congestive heart failure, vascular thrombosis and/orembolism, chronic obstructive pulmonary disease, asthma, hemolyticanemia, G6PD and 6PGD deficiency in RBC, sepsis, hemorrhagic shock, andinfant jaundice.

Aspect 4 provides a composition comprising one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, and hydroxypyruvatic acid and salts and esters thereoffor use in a method of treating or preventing a disease or disorderaccording to aspect 2, wherein the metabolic syndrome is selected fromthe group of diabetes, diabetic neuropathy, and diabetic nephropathy.

Aspect 5 provides a composition comprising one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, and hydroxypyruvatic acid and salts and esters thereoffor use in a method of treating or preventing a disease or disorderaccording to aspect 2, wherein the disorder associated with metabolismis selected from the group consisting of mitochondrial DNA depletion andother mitochondrial diseases, Leigh syndrome, epilepsy, bipolardisorder, psychiatric disorders and mood disorders, cerebrovascularaccident, damage from acute head injury, acute or chronic renal failure,acute or chronic liver failure, splenomegaly, acute or chronicpancreatic failure, chronic auto inflammation and autoimmune syndromeand diseases, psoriasis, impairment in collagen synthesis,osteoarthritis, pre-eclampsia, thyroid disease, chronic fatigue, andfibromyalgia.

Aspect 6 provides a composition comprising one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, and hydroxypyruvatic acid and salts and esters thereoffor use in a method of treating or preventing a disease or disorderaccording to aspect 2, wherein the degeneration disease related to theaging process of an organism, or the degeneration disease acceleratingthe aging process of an organism is selected from the group consistingof age related hearing loss such as presbyacusis, noise induced hearingimpairment or ototoxic hearing impairment, age related maculadegeneration, glaucoma, optic neuropathy, ischemic optic neuropathy,retinitis pigmentosa, osteoporosis, chronic neurodegeneration,amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease,multiple sclerosis, Huntington's disease, and prion disease.

Aspect 7 provides use of a composition comprising one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, and hydroxypyruvatic acid and salts and esters thereoffor enhancing physical training, performance and recovery from exercise.

Aspect 8 provides a composition comprising one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, and hydroxypyruvatic acid and salts and esters thereoffor use as a medicament having an antioxidant activity via enhancingendogenous antioxidant protection of living cells, tissues and/or wholeorganisms.

Aspect 9 provides a use of a composition comprising one or morecompounds selected from the group consisting of D-glyceric acid,DL-glyceric acid, L-glyceric acid, and hydroxypyruvatic acid and saltsand esters thereof for increasing the muscle yield and simultaneouslydecreasing of fat content of a human or an animal, and/or decreasingnutrition consumption without losing muscle mass of an animal, such as amammal, poultry, and fish.

Aspect 10 provides a composition comprising one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, and hydroxypyruvatic acid and salts and esters thereoffor use in a method of treating or preventing a disease or disorderaccording to any one of aspects 1 to 6, or enhancing physical training,performance and recovery from exercise according to aspect 7, or for useas a medicament according to aspect 8, or for use according to aspect 9,wherein the composition further comprises a pharmaceutically acceptableexcipient.

Aspect 11 provides a composition comprising one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, and hydroxypyruvatic acid and salts and esters thereoffor use in a method of treating or preventing a disease or disorderaccording to any one of aspect 1 to 6, or enhancing physical training,performance and recovery from exercise according to aspect 7, or for useas a medicament according to aspect 8, or for use according to aspect 9,wherein the composition is in a form of a solution, syrup, powder,ointment, mixture, capsule, tablet, or an inhalable preparation.

Aspect 12 provides a composition comprising one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, and hydroxypyruvatic acid and salts and esters thereoffor use in a method of treating or preventing a disease or disorderaccording to any one of aspect 1 to 6, or enhancing physical training,performance and recovery from exercise according to aspect 7, or for useas a medicament according to aspect 8, or for use according to aspect 9,wherein the composition is in a form suitable for parenteral, oral,topical or inhalable administration.

Aspect 13 provides a composition comprising one or more compoundsselected from the group consisting of D-glyceric acid, DL-glyceric acid,L-glyceric acid, and hydroxypyruvatic acid and salts and esters thereoffor use in a method of treating or preventing a disease or disorderaccording to any one of aspect 1 to 6, or enhancing physical training,performance and recovery from exercise according to aspect 7, or for useas a medicament according to aspect 8, or for use according to aspect 9,wherein the composition is part of a beverage, a food product, afunctional food, a dietary supplement, or a nutritive substance.

Aspect 14 provides a pharmaceutical composition comprising an effectiveamount of one or more compounds selected from the group consisting ofD-glyceric acid, DL-glyceric acid, L-glyceric acid, and hydroxypyruvaticacid and salts and esters thereof for use according to any one ofaspects 1 to 13.

Aspect 15 provides a method of increasing direct or indirectmitochondrial activity, RNA expression of genes encoding ETS relatedgenes, TCA activity, and/or biogenesis of new mitochondria in a subjectcomprising administering a composition comprising an effective amount ofone or more compounds selected from the group consisting of D-glycericacid, DL-glyceric acid, L-glyceric acid, hydroxypyruvatic acid and saltsand esters thereof to a subject in need.

Aspect 16 provides a method of treating or preventing a disease ordisorder in a subject comprising administering a composition comprisingan effective amount of one or more compounds selected from the groupconsisting of D-glyceric acid, DL-glyceric acid, L-glyceric acid, andhydroxypyruvatic acid and salts and esters thereof to a subject in need.

Aspect 17 provides a method according to aspect 16, wherein the diseaseor disorder is as defined in any one of aspects 1 to 6.

Aspect 18 provides a method of enhancing physical training, performanceand recovery from exercise, or reducing radical oxygen species withantioxidants in a subject comprising administering a compositioncomprising an effective amount of one or more compounds selected fromthe group consisting of D-glyceric acid, DL-glyceric acid, L-glycericacid, and hydroxypyruvatic acid and salts and esters thereof to asubject in need.

Aspect 19 provides the method according to any one of aspects 15 to 18,comprising administering the composition comprising one or morecompounds selected from the group consisting of D-glyceric acid,DL-glyceric acid, L-glyceric acid, hydroxypyruvatic acid and salts andesters thereof, and a pharmaceutically acceptable excipient.

Aspect 20 provides the method according to any one of aspects 15 to 19,comprising administering the composition in a form of a solution, syrup,powder, ointment, capsule, tablet, or an inhalable preparation.

Aspect 21 provides the method according to any one of aspects 15 to 20,comprising administering the composition via a parenteral, oral, ortopical application route or by inhalation.

Aspect 22 provides the method according to any one of aspects 15 to 21,comprising administering the composition via a beverage, a food product,a functional food, a dietary supplement, or a nutritive substance.

The present invention is illustrated by the following non-limitingexamples. The examples constitute an entirety of findings from varioustissues, organs, and whole physiological system from humans and animalsin different metabolic states or dosing etc. supporting each other.

EXAMPLES Example 1

The purpose of first 4 separate in vitro studies with primary humanhepatocytes (studies 1-4 in Example 1.1) was to investigate the effectof D-glyceric acid, calcium salt dehydrate (product number:367494/Sigma-Aldrich, later also DGAcs) to the cell viability andcellular reactive oxygen species (ROS). Also HPA and LGA were tested insome experiments.

Additional 2 studies (5-6 in Example 1.2) were conducted in order toverify and specify results from first 4 studies. On top of viability(LDH) and ROS analyses also gene expression and NAD⁺/NADH-ratio wasmeasured from human primary hepatocytes in studies 5-6 in Example 1.2.

Furthermore in studies 5-6 the accuracy of cell viability results basedon LDH method was double checked with independent estimate on viabilityusing the gene expression of the so called housekeeping genes from thecell cultures. The results of these independent viability tests werevery well in line confirming that mostly used LDH estimation methodworked well, and also that results from gene expression analyses areconsistent in respect to dose responses etc.

Validity of the used ROS estimation method was checked by using knownantioxidants treatments that are known to decrease ROS as a positivecontrol of the method. Used ROS estimation method gave very reasonableresults with e.g. vitamin C, vitamin E, glutathione, and alfa-lipoicacid.

The age of the human donors in these in vitro studies 1-6 varied between47-65 years. All had some kind of medical history and specified medicalcause of death, i.e. they were not healthy volunteers as was mostly thecase in clinical in vivo experiments, see Examples 2.1-2.4.

Example 1.1 Measurement of LDH and ROS from Human Primary Hepatocytes

Materials and Methods

Primary human hepatocytes were purchased from Celsis In VitroTechnologies (1450 South Rolling Road Baltimore, Md. 21227, USA).Primary hepatocytes from altogether 4 donors aged 47 (YJM, female), 57(DOO, male), 58 (CDP, male), and 54 (JGM, female) were used. Accordingto the information provided by the Celsis, hepatocytes from each donorshould have at least 70% viability and more than 5 million viable cells.The medium for the culture of hepatocytes was provided by Celsis. Theywere InVitroGRO CP (for plating) medium (Z99029) and InVitroGRO HI (forincubation) medium (Z99009). Antibiotics (Torpedo Antibiotics Mix,Z990007) were also from Celsis. The thaw, plating and culture of cellswere carried out according to the instruction provided by the Celsis InVitro Technologies.

The other reagents for experiments were D(−) fructose (Sigma-Aldrich,F0127), D(+)-glucose (Sigma-Aldrich G7528), Dulbecco's phosphatebuffered saline (DPBS) (Lonza, BE17-512F), absolute ethanol (ProLab20821.365) and foetal bovine serum (Thermo Fisher, SV30160). For thecell culture, BD BioCoat™ Collagen I Coated 96-well Black/Clear Plates(354649) were used. Other plastic ware used in this study was purchasedfrom Sarstedt Ltd (Leicester LE4 1AW, UK). Cells were cultured in a cellculture incubator (Sanyo MCO-18AIC) at an atmosphere of 37° C. and 5%CO₂.

For the measurement of cell viability after treatment of test compound,CytoTox-One Homogeneous Membrane Integrity Assay kit (Promega, G7891)was used. The CytoTox-One Assay is a rapid fluorescent measure of therelease of lactate dehydrogenase (LDH) from cells with a damagedmembrane. The number of viable cells correlates to the fluorescenceintensity determined by a fluorescence plate reader (Hidex Chameleon Vmultiplate reader, Hidex Oy, Turku, Finland) with excitation 544 nm andemission 590 nm. For the measurement of cellular reactive oxygen species(ROS), DCFDA Cellular ROS Detection Assay Kit from Abcam (ab113851) wasused. Reactive oxygen species (ROS) assay kit (ab113851) uses the cellpermeant reagent 2′,7′-dichlorofluorescein diacetate (DCFDA), afluorogenic dye that measures hydroxyl, peroxyl and other ROS activitywith the cell. The activity of ROS was measured by a fluorescence platereader (Hidex) with excitation/emission wavelengths of 485 nm/535 nm.

This study was conducted in accordance with the relevant standardoperating procedures (SOPs) in BioSiteHisto Oy.

Cell Culture

The culture of primary hepatocytes was carried out based on theinstructions provided by Celsis. After thawing, the cells were suspendedin InVitroGRO CP medium. Thereafter the viability of cells wasdetermined using the Trypan Blue exclusion method. Then theconcentration of cells was adjusted using InVitroGRO CP medium, so about30000-35000 cells/100 μl/well can be seeded in collagen I coated 96 wellplate (BD, 734-0248).

After seeding, the cells were cultured overnight in the cell cultureincubator (Sanyo MCO 18) at atmosphere of 95% air, 5% CO₂ at 37° C.Thereafter, the culture medium for the cells was changed to InVitroGROHi (Hi Medium) containing test compound (D-glyceric acid calcium saltdehydrate) at the concentrations of 0, 0.2, 2 and 20 μg/ml (study 1,Table 1.1.1) and 0, 0.4, 2 and 10 μg/ml (study 2, Table 1.1.2).

In study 3 also LGA and HPA were tested and compared to DGA (see FIGS.11c and 11d ) with same doses as in study 2.

In study 4 DGA (molecular weight=106 g/mol) was tested in equimolardoses against other substances with known antioxidative properties, i.e.vitamin E (trolox, T3251 Sigma, molecular weight=431 g/mol), glutathione(G6013 Sigma, molecular weight=307 g/mol), vitamin C (L-ascorbic acid,A4544 Sigma, molecular weight=176 g/mol) and morin dehydrate (M4008Sigma, molecular weight=302 g/mol). The results from study 4 arepresented in FIGS. 6a and 6 b.

In studies 2-4 additional diet with 0.75 mM of palmitic acid (“fattyacid diet”) was added to Hi Medium. Hi Medium in itself contains ampleamounts of glucose and fructose and other necessary ingredients forkeeping viability of the cell culture at optimal level.

After 24 h (in study 1) and 20 h (in studies 2-4) incubation medium wasrenewed. Second renewal of the incubation medium occurred at 48 h instudy 1 and at 40 h in other studies. ROS and LDH were measured after1.5-2 hours after last change of the medium, i.e. in a situation withmoderate metabolic stress.

LDH Measurement

The measurement of LDH from medium (leaked LDH) and from cell and medium(total LDH) was carried out according to the instruction provided by thePromega (G7891). The plate was incubated at an incubator to achieve atemperature of 22° C. Thereafter, equal volume of CytoTox-ONE Reagent tocell culture medium (100 μl) was added to each well and mix for 30seconds. Then they were incubated for 10 min at 22° C., and then 50 μlof Stop Solution was added to each well. After gentle mixing, thefluorescence signal was measured at an excitation wavelength of 560 nmand emission wavelength of 590 nm using the Hidex Chameleon V multiplatereader (Hidex Oy, Turku, Finland).

For the total LDH measurement, all the steps are same except a 2 μl ofLysis Solution (per 100 μl original volume) will be added to the eachwell to lyse the cells before CytoTox-ONE Reagents will be added.

Cellular ROS Detection

The measurement of cellular reactive oxygen species (ROS) was carriedout according to the instruction of Abcam (ab113851). At the end oftreatment time point, culture medium was taken away for leaked LDHmeasurement and cells were washed with 100 μl/well PBS once. Thereafter100 μl/well of DCDA mix was added and incubated for 45 min at 37° C. inthe dark. Then cells were washed once with 1× buffer solution. Thefluorescence signal was measured at an excitation wavelength of 485 nmand emission wavelength of 535 nm using the Hidex Chameleon V multiplatereader (Hidex Oy, Turku, Finland).

Calculation and Interpretation of Results

For the LDH and ROS assays, individual values of each measurement werestored and average value of fluorescence signals of various repeats ofeach treatment at studied time point were calculated and compared. AllROS calculations possessed 6 repeats for all measurement point.Viability (LDH) measurements contained 4-6 repeats depending on thestudy. Results are presented in FIGS. 6-11. In study 1 (48 h &YJM andDOO) not all the data points for both donors were measured. Those pointsare marked by not available (n.a.).

Results from Studies 1-4

From FIGS. 5-11 it can be concluded that in standard environment andtreatment, i.e. no excess nutrition or starvation DGAcs and also LGA andHPA can modestly increase the viability of human primary hepatocytes (orin any case at least keep it stable). Altogether four donors, two donorsfrom both sexes, were tested.

More importantly DGAcs and also HPA and LGA can decrease ROS levelssignificantly compared to 0 controls. In used cell model ROS levels arecalculated under moderate metabolic stress meaning that measurements aremade 1.5-2 hours after the change of the medium, i.e. after givinghepatocytes fresh medium with new nutrition. In study 4 with CDP (maledonor, in FIG. 5) and with JGM (female donor), it was shown that two day(48 h+1.5 h) administration of DGAcs decreases ROS significantly.Furthermore this decline is at least as large as with other known strongantioxidants (vitamin E, glutathione, vitamin C, and morin dehydrate).DGA seems to work both in moderate metabolic stress induced by glucose(Hi Medium only) (FIG. 6a , with JGM) and also in moderate metabolicstress induced by beta oxidation (Hi Medium+0.75 mM of palmitic acid)(FIG. 6b , data from JGM). With female donor YJM and in HiMedium+Palmitic acid LGA and HPA doses of10 μg/ml reduce the level ofROS by 30-35% compared to control (FIG. 11c ). DGA gave same decline inwith JGM (also a female donor) with same conditions (FIG. 11a ). ROSdeclined significantly with DGA compared to 0 doses also with otherdonors as well.

Consistent decline in ROS seen in multiple studies is a clear indicationand a follow up of activation Nrf2/ARE antioxidant defense system by theuse of DGAcs.

Statistical relevance of each result can be estimated using standarddeviations (std) calculated for each study point. Deviations+/−std OrSEM from calculated averages are presented for all study points(additional+/−line segment on each bar). (In FIG. 6-10 the standarddeviations are calculated from individual observations and in FIGS. 5and 13-14 standard error of the mean (SEM). SEM is otherwise the same asstd but it is divided by square root of the number observations (N).).Statistical significance is indicated by * and ** marks (*=P-value<0.05, and **=P-value <0.01).

In some exceptional cases the hepatocytes viability in vitro hasincreased even by 40-60% and notably in the same test setting, with 0.4DGA dose, also statistically significant decrease in viability wasobserved (FIG. 10c ). Strong reactions clearly indicate that DGA hassignificant impact on the activity of the hepatocytes. Additionally,strong viability or cytotoxity effects seen in FIG. 10c can beinterpreted also as increased signaling for apoptosis and/or cellsurvival, i.e. increased control of cell cycle.

When combining the above viability observations to the observations thathepatocytes kept under calorie restriction (CR=no addition of food, i.e.no change of medium during 48+1.5-2 h) died clearly more likely thancontrol (FIGS. 10a and 10b ), it seems obvious that metabolic flux hasbeen increased by the use of DGAcs. Furthermore it seems also that invitro experiments the cells do not have the means to balance significantimpact from DGA use like in vivo. In case of starving diet (CR), theobvious signal for increased apoptosis was the limited amount ofnutrition like seen also in below examples with DOO and YJM.

From FIGS. 9a-d it can be seen that the increase in viability in HiMedium for DOO is statistically significant but for YJM it is not. FromFIG. 10c it can be seen that the increase of viability for CDP due toDGA is statistically very significant. Furthermore from FIGS. 10a and10b it can be seen that starving diet increases cell death the higherthe DGA dose has been. This deviation from 0 control is statisticallyvery significant for doses DGA 2 (DOO) and DGA 20 (YJM).

Enhanced control of the cell cycle increases the protection againstdeveloping cancer. In combination with enhanced control and activationof intracellular metabolic processes, it also decreases the risk of anonset of several other diseases, including but not limited toauto-inflammatory and auto immune diseases.

Significant decrease in ROS levels, i.e. a clear increase in endogenousdefense against oxidative stress, alleviates and prevents the onset ofseveral or even all degenerative diseases (and also cancer). Decrease inROS in cardiovascular system reduces the risk of cardiovascular diseasese.g. atherosclerosis.

Example 1.2 Measurement of NAD⁻/NADH -Ratio, Viability and ROS fromHuman Primary Hepatocytes

In studies 5 and 6 same donors were used as in Example 1.1. Cellculturing was according to standard protocols (24 h cycle) and Hi Mediumwas used like in most experiments 1-4. The 3 donors chosen were DOO(male), JGM (female), and CDP (male). By using same donors we coulddouble check methods used.

Viability and ROS confirmation: Without going into details for repeatedviability and ROS results presented already in Example 1.1, we justconclude that DGAcs and also HPA reduced ROS compared to control, andthat viability measurement using housekeeping gene expression confirmedLDH viability results.

Example 1.2.1 NAD⁺/NADH-Ratio in Human Hepatocytes

For the measurement of total NAD (NAD_(tot)=NADH+NAD⁺) and NAD⁺/NADHratio, a NAD⁺/NADH Quantification kit (MAK037) from Sigma was used. NADfunctions as an electron carrier, fluctuating between the oxidized(NAD⁺) and reduced (NADH) forms. In addition, NAD⁺ plays critical rolesin ADP-ribosylation reaction and as a substrate for sirtuins. Accordingto the Sigma instruction, the NAD⁺/NADH Quantification kit (Sigma)provides a convenient tool for sensitive detection of NAD, NADH andtheir ratio without requirement to purify them from samples. Importantto notice is that the kit measures NADH and NAD⁺ in whole cell includingall cell organelles and compartments. Mitochondrial matrix is clearlydominant NADH producer from TCA and beta oxidation (consuming naturallythe same amount of NAD⁺ in redox reactions). Importantly, formed NADH isalso typically oxidized into NAD⁺ in the matrix by Complex I in ETC. Asis well known anaerobic carbohydrate metabolism, i.e. glycolysis, incytosol also consumes NAD⁺ and produces NADH.

Cell culture in NADH -tests was like in Example 1.1. The change of themedium with DGAcs was conducted at 0 h, 24 h and 48 h. NAD measurementswere made at 48+3 h in all cases. For CDP also HPA was tested, and forDOO no new nutrition at 48 h was added, i.e. test was conducted infasting conditions. Results are presented in table 1.2.1 below.

TABLE 1.2.1 Effect of DGAcs and HPA on NAD⁺/NADH-ratio at 48 h + 3 hTreatments DGAcs DGAcs Donor/measurement Control 1.4 14 HPA 14 JGM (newnutrition at 48 h), N = 5 NAD_(tot) 0.345 0.329 0.302 NADH 0.280 0.2890.294 NAD+ 0.064 0.040 0.009 NAD+/NADH 0.229 0.139 0.029 Change inNAD+/NADH vs. −39.3% −87.2% Control P-value  4.3%  0.27% DOO (no newnutrition at 48 h), N = 3 NAD_(tot) 0.238 0.243 0.240 NADH 0.227 0.2390.232 NAD+ 0.011 0.004 0.009 NAD+/NADH 0.049 0.017 0.037 Change inNAD+/NADH vs. −64.6% −24.0% Control P-value   2.7%  24.5% CDP (newnutrition at 48 h), N = 5 NAD_(tot) 0.235 0.231 0.232 0.232 NADH 0.2170.222 0.225 0.222 NAD+ 0.018 0.009 0.007 0.010 NAD+/NADH 0.085 0.0400.032 0.045 Change in NAD+/NADH vs.   −53%   −62%  −47% Control P-value 0.25%  0.15% 0.12% Donor/measurement Control DGAcs DGAcs HPA 14 1.4 14P-values are presented in the table

As one can see from table 1.2.1, DGAcs administration clearly decreasesNAD⁺/NADH-ratio at the cellular level as a whole. There is also a cleartendency for all donors that NADH levels increase with DGAcs compared tocontrol. Increased NADH level is a sign that the aerobic ATP energycreating capacity of the hepatocytes is increased. We can indicativelyalso conclude that ATP production (and thus also its consumption) isincreased. This is consistent with the observation of increasedapoptosis (cell death) in starving diet compared to normal feeding inhepatocytes (clear result seen in Example 1.1). Cell death in starvingdiet is caused by increased energy consumption by the hepatocytes andthis leads to increased cell death in starving diet conditions.

Cells have to produce ATP all the time for sustaining normal cellfunctions, but they increase ATP production only, if it is consumed tosome task, e.g. physical exercise or thermogenesis. In cell culture atcontrolled 37° C. there is no need for increased thermogenesis. Neitheris there any physical exercise. The probable use of formed extra ATPenergy is very likely due to anabolic reactions, e.g.gluco-/glyceroneogenesis and protein synthesis that consume a lot ofenergy (see FIGS. 3a and 3b , and Example 4). Also increased control andcorrection of anabolic processes like protein synthesis and enzymeformation in the ER consume energy (e.g. ATP-dependent chaperones).Increased supply of substrates, increase in pyruvate and aminogroups(=decrease in urea cycle), for anabolic reactions by the DGAcsadministration (shown in Example 2.1-2.3) supports also the idea thatexcess ATP is consumed for enhanced renovation of enzymes and similarcomplex macromolecules. In general these processes that increase ATPconsumption are beneficial to cells and promote long term health.

Interestingly the results in Table 1.2.1 also show that NADH+NAD⁺ levelsaltogether do not increase as much as NADH. Mathematically this meansthat the NAD⁺ level decreases in DGAcs groups. This is naturally mostlydue to NAD⁺ reduction into NADH but also increased NAD⁺ consumption bysirtuins e.g. NAD⁺ dependent deacetylation of PGC-1a can be oneconsistent explanation. From other examples (see below) we can clearlysee that the use of the DGAcs induces increase in aerobic metabolism andalso mitochondrial biogenesis, and it is well known that deacetylationof PGC-1a activates a flow of genes that up regulate aerobic metabolism.(From human in vivo leukocyte gene expression we can see that PGC-1a andrelated energy metabolic genes are up regulated by DGAcs.)

Importantly, this is consistent with the notion that cytosolic NAD⁺generating capacity of the cells increases by the DGAcs administrationand that at the same time DGAcs decreases NAD⁺ levels as a whole inaerobic cells. In fact the use of DGAcs clearly increases cytosolic NAD⁺generating capacity by increasing MA-shuttle intermediates (due tosignificant pyruvate increase, see Example 2.3) and also due to increaseof GP-shuttle formation reflected by increased gene expression of GPD2,in Example 2.2. (GPD2 up regulation is also very likely due to increasedsubstrate formation by mitochondrial beta oxidation. Liberating fattyacids for beta oxidation increases intracellular concentration of freeglycerol, which is phosphorylated by kinase enzymes (located on theouter mitochondrial membrane) into G-3-P (see FIG. 1b and Example 4below). Increased activity of MA- and GP-shuttles by the Inventionincreases cytosolic NAD⁺ levels when needed and clearly more rapidlycompared to without using the DGAcs. Cytosolic NAD⁺ must be generatede.g. in order to keep the flow of glycolysis active. Otherwise there isa risk of generating AGEs (advances glycation end products). AGEs, aresubstances that can be a factor in the development or worsening of manydegenerative diseases, such as diabetes, atherosclerosis, chronic renalfailure and Alzheimer's disease

Example 1.2.2 Gene Expression from Human Hepatocytes

For DOO, JGP and CDP also gene expression analyses was made. In the usedtechnology (see Example 2.3.3 for details) the expression of so calledhousekeeping genes is measured as well as the expression of someselected genes. The expression of the housekeeping genes, by definition,is always high. The results from their measurements from hepatocytelysates were volatile but statistically meaningful. Housekeeping geneexpression indicated very similar changes in viability that also LDHstudies had pointed out earlier.

TABLE 1.2.2 Indicative gene expression results from hepatocytes lysatesGene expression deviation from control, % increase or decrease HPA DGAcs1.4/ DGAcs 14/ 14/ Donor/Gene Control Control Control JGM, N = 5 HO-154% 50% n.a. CYP2B6 8% 3% n.a. PGC-1a 12% 42% n.a. DOO, N = 5 HO-1 83%21% 1% CYP2B6 29% 49% 7% PGC-1a 45% 1% −2%  CDP, N = 5 HO-1 n.a. n.a.n.a. CYP2B6 38% 45% n.a. PGC-1a 5% 20% n.a. Combined p-value <0.05 forHO-1 and CYP2B6, for PGC-1a P-value was <0.10

For combined data in DGAcs 1.4 μM group vs. the control both HO-1 andCYP2B6 were statistically significantly different from control (P-valueis approximately 1%, i.e. the result was statistically verysignificant). Combined test for PGC-1a yielded P-value of some 10%,which indicates that also PGC-1a was activated compared to control.Importantly the deviations from relevant controls were in line with invivo results with leukocytes (table below and Example 2.3.3).

We can conclude that there is a clear tendency for e.g. inducible hemeoxygenase (HO-1) to rise in hepatocytes after the use of the DGAcs (anindication of Nrf2/ARE activation), and that the expression of themaster regulatory gene of energy metabolism, PGC-1a seems to rise inhepatocytes also (see FIG. 2 for the relevance of these genes). Evenmore convincing evidence on the ability of the use of DGAcs to activatethese pathways and also e.g. on CYP2B6 is presented in the next example,the in vivo clinical experiment. Interestingly inducers of CYP2B6 areused in e.g. as anti-epilepsy drugs and mood stabilizers, e.g.management of Bipolar Disorder.

Example 2

In Vivo Clinical Studies

Altogether three short 1-4 day in vivo clinical tests with varying doseswere conducted, and additionally several longer term tests ranging from11 day to 8 weeks (Example 2.1). Additionally, after received full proofof efficacy and safety, also higher and also acute doses were tested(Example 2.2). The doses used in these clinical tests are at maximumless than 10% of the safe doses with rats in the below described 3 weektests.

In the first set of experiments presented in Example 2.1 all the bloodmeasurements were conducted next day after last administration of DGAcs.In the longer experiments doses (3-5 mg/kg) were typically taken once aday (preferably in the evening before going to bed). In shorter 1-4 dayexperiments doses were taken twice a day, in the morning and beforegoing to bed. In all experiments in Example 2.2 the last DGAcs dose wasdouble in size (some 8 mg/kg), and it was taken in the same morning asthe collection of blood samples. Relatively high dose and short time tomeasurement was chosen in order to see clearer dose response in geneexpression from peripheral leukocytes, and also from collected plasmasamples.

Example 2.1: First Set of Clinical In Vivo Experiments

Eight (8) persons completed controlled clinical testing with standard10-12 hour fasting diet (f) blood test (sample analytics by United MedixLaboratories, Finland). There were four different types of clinicaltests: first a 3 week test with low dose of DGA (N=3), secondly one 11day test with bigger dose (N=1), and finally a 4 day test with high dose(N=4) and with low dose of DGA (N=2). Subjects 1 and 4 (in table 3) didthe first three week test and then some months later also the separate 4day test.

Daily doses varied from 3-4 mg/kg/day in 3 week test to 6 mg/kg twice aday in 4 day test. General rule in the studies was that the shorter theperiod the higher the dose. In three week clinical tests fasting bloodstandard lipid panel and other basic readings were measured at thebeginning and at the end of the test period.

In the 4 day tests very wide fasting blood panel consisting of 25different metrics was measured (see table 3 below), and additionally forsome participants full blood count to measure more precise effect onerythrocytes and hemoglobin was carried out.

Unlike in hepatocytes study (Example 1) clinical experiments wereconducted in a fasting diet situation. Challenges for obtainingmeaningful health indications from DGAcs administration arises from thefact that the study persons (aged 41-73) were all healthy volunteers,i.e. the blood values were mostly at very good levels, and thusimprovements from the control are hard to achieve. Nevertheless theresults from clinical testing show clear signs on the efficacy of DGA inimproving systemic redox state of especially the cardiovascular systemand also clear indications of increased overall metabolic flux. Specificmarkers for functioning of liver, kidneys, pancreas and spleen showimprovement in general (see Table 4). The results from 10-12 hourfasting blood test after 4 day administration for lean and healthyvolunteers are presented in Table 3.

TABLE 3 Four day human trial with 5-6 mg/kg/twice a day. DGA calciumsalt was mixed to 1 dl of water in advance. Healthy subjects with BMI<24.9, i.e. normal weight/lean. (N = 4) Measurements on Monday andFriday morning, standard fasting blood test. 4 day changes from zerocontrols, % S1 S2 S3 S4 S-Afos −2.1% 0.0% −5.8% −6.3% S-alat −13.6%−13.3% 57.8% 11.1% S-Alb −2.2% 0.0% 0.0% −2.2% S-Amyl 64.9% 6.3% −6.7%−73.8% S-Asat −33.3% −40.9% −7.7% −4.3% S-Bil −19.1% −22.8% −58.1%−12.6% S-Bil-Kj −17.4% −23.3% −58.4% −8.8% S-Cal −1.7% 0.0% −2.2% −2.5%S-CK −21.7% −24.7% −65.9% 3.8% fS-Fe −27.9% −22.5% −72.0% −5.2% fS-Gluk1.9% −9.8% −8.9% 0.0% S-GT 11.1% 22.2% −9.5% −6.7% S-K −2.3% 10.8% 7.0%−4.7% fS-Kol −1.6% −5.7% −6.4% −3.2% HDL −4.5% −3.3% −9.1% −5.9% LDL2.4% −10.7% −5.3% −9.5% fS-Krea −4.4% −11.1% −5.0% −8.5% S-LD (LDH)−3.7% −4.2% n.a. −4.8% S-Mg −2.4% 6.3% −3.6% 2.2% S-Na −1.4% −2.1% −2.1%0.7% fS-Pi 18.3% 9.3% −6.0% −6.4% fS-Transf 10.0% 0.0% 0.0% −4.2%fS-Transferr.satur. −32.3% −23.3% −72.4% 0.0% fS-Trigly −4.6% 39.7%35.4% 50.4% S-Uraat −5.2% −3.2% −12.0% −9.9% fS-Urea −7.1% −15.9% 12.8%−7.3%

Longer run 3 week tests with 3-4 mg/kg/day dose were conducted withsubjects S1 and S4 (from Table 3), and additionally on subject S6.Similar 11 day test with 2×4 mg/kg/day dosing was carried out for S5. Inthese tests only fS-Kol, fS-Trigly, fS-Glucose, fS-Krea and fS-GT weremeasured (and blood pressure for S4 and S5, FIG. 12). Subjects S7 and S8did also 4 day trial but with only 2×3 mg/kg/day dose and did not bringany significant changes in observed 25 blood metric (in Table 3) exceptpossibly with slightly increased blood triglycerides (fS-Trigly) andlowered blood uric acid levels (S-Uraat).

Putting S1-S6 results together like in Table 3, one can observe cleartendency towards lower cholesterol (fS-Kol) for all subjects, and sometendency towards lower sodium (S-Na) and glucose (fS-Glucose) levels forall. Creatinine kinase (S-CK) was on average clearly lowered after theuse of DGAcs.

Putting S1-S8 together one can observe some tendency for increase inblood triglycerides level. This was not the case for 2 out of 8 subjectsbut for 5 subjects the increase was in the range of 25-50% percent.

When interpreting the significance of the results from healthy volunteertesting, it should be noted that the study subjects S1-S4 in Table 3 hadtheir blood counts at relatively optimal levels before DGAadministration. As an example blood glucose for all subjects was in therecommended range of 4.2-6.0 mmol/l. Specifically subjects S1 (5.2mmol/l) and S4 (4.7 mmol/l) in Table 3, whose blood glucose did notdecline due to DGA administration, had no physiological need forreduction in fasting state blood glucose concentration. Also S-Asat,S-Bil, S-Bil-Kj, S-Fe, fS-Krea, S-Na and S-Uraat levels were at normalranges for all subjects. For more information and interpretations ofvarious metrics see table 4 below.

In a summary, already in 4 days surprisingly big changes in abovedescribed blood metrics can be observed, and they are basically alwaysto a direction indicating improved health from the use of DGAcs. This isremarkable and proves the enhancements achieved with DGAcsadministration in 1) the redox-state of the cells, i.e. by re-oxidizingNADH+H⁺ to NAD⁺, 2) in the velocity ATP production (and metabolic fluxof sugars and fats), 3) antioxidative state of the cells, e.g. byhindering excessive radical oxygen species (ROS) formation fromoxidative phosphorylation (OXPHOS) and, 4) protein synthesis and enzymeassembly, especially seen (but not limited to) in assembly of heme (Fe)containing enzymes typically related to oxidative metabolism. (IncreasedFe use for proteins is seen particularly in next example (2.2) in whichHO-1 enzyme activity and related catabolism of heme into biliverdin andfurther to bilirubin+Fe+CO (see FIG. 4) has been increased by higher andmore acute DGAcs dose, and despite this increase of free Fe output, theFe concentration in blood decreases in healthy volunteers.)

Example 2.2 Extended Administration and Control Period for S4

For Subject 4 (S4) in Example 2.1, the 4 day test period was prolongedby 8 weeks with reduced, only once a day 5 mg/kg/day administrationbefore going to bed. Additionally in order to have an understanding thatthe results in the 8 week test were due to the administration of theInnovation, an additional negative control measurement without DGAcsafter 2 weeks and 2 days was made (+2 days due to alleviating doses seebelow). The idea was to see, if the beneficial effects seen in 4 daytreatment prevailed in longer term (positive control).

One additional point of interest was to check that whether the elevatedblood triglycerides come down from slightly elevated levels after first4 days. Blood triglycerides seem to increase for most of the testedsubjects in the short run compared to the controls. This increase islikely mostly due to the increased de novo biosynthesis of fatty acidsand triglycerides by the liver for increased mitochondrial betaoxidation (see FIG. 3a and Example 3). De novo synthesis produces mostlymedium chain fatty acids that are easily metabolized e.g. in betaoxidation and are considered to possess even health effects, and thusthe observed increase in fS-Triglys can be health promoting.

TABLE 2.2.1 Follow up test, 8 week positive control, followed by 2 weeknegative control. DGAcs was mixed to water in advance. (N = 1, healthyfemale, age 52 years) Measurements in the mornings at 07:35-07:50,standard fasting blood test, changes from previous observation, % S4 S4(2 week S4 (after 8 weeks) follow up) (4 day test) Positive ControlNegative Control 0 day 4 day +8 weeks +2 weeks S-alat, change 11.1%85.0% −35.1% S-Asat, change −4.3% 31.8% 24.1% AST/ALT 1.28 1.10 0.781.44 S-Bil 11.1 9.7 7.2 10.2 S-Bil, change −12.6% −25.8% 41.7% fS-Fe21.3 20.2 15.6 15.1 fS-Fe, change −5.2% −22.8% −3.2% fS-Trigly 1.15 1.731.35 1.11 fS-Trigly, 50.4% −22.0% −17.8% change S-Uraat 273 246 247 295S-Uraat, −9.9% 0.4% 19.4% change

In Table 2.2.1 only some results from the blood samples are presented.AST/ALT: the tendency for all tested of an improvement in AST/ALT-ratioseems to prevail very clearly also in the longer term, and importantlyAST/ALT returns back to starting levels 2 weeks after stopping theadministration. Bilirubin: the tendency for all tested for a reductionin blood bilirubin seems to prevail very clearly also in the longerterm, and importantly they return back to starting levels 2 weeks afterstopping the DGAcs administration. These results with bilirubin (andHO-1) are very interesting because, as seen in next Example 2.3, HO-1enzyme can be activated and increase bilirubin production, which is thefinal output from heme degradation to biliverdin. Blood iron (Fe): Fecontinued to decline during the 8 week period and reached the samemagnitude of decline as for other subjects in already 4 days (table 3).For some unknown reason Fe did not return to levels prevailing beforethe experiments. fS-Trigly: As seen from Table 2.2.1, triglycerideslevel drops significantly in 8 weeks from elevated level after 4 dayadministration. Interestingly the level of 1.35 seems to remain atslightly higher levels than without the use of DGAcs(=1.11-1.15).Slightly elevated blood triglycerides in humans are probably anindication on increased mitochondrial beta oxidation (like seen inexample 3 with rats). S-Uraat: down with DGAcs in the short and longerrun. Back to original levels after stopping the administration.

Withdrawal symptoms: Maybe the most important result from this 8 weekfollow up test with S4 was seen when stopping daily 5 mg/kg/dayadministration. After 36 hours without DGAcs dose, negative symptoms ofthe “withdrawal” started. They were very clear but not severe includingbad feeling inside the head and overall dizziness and uneasiness. It wasdecided that S4 can receive some alleviating small doses of DGAcs, andthat the 2 week test period without the use of the DGAcs is postponed bythese days. First alleviating dose (3 mg/kg) was received after 48hours. The “balancing” effect was felt very fast. Already in 30 secondto one minute general feeling of S4 returned more or less to normal.Fast alleviating physical effect was likely related to strong signalingto the body that the beneficial substance for energy metabolism andoxidant defense is still available (see FIG. 1.b description). Anotheralleviating dose of only 1.5 mg/kg was taken during the next day, andthere after started the 2 week negative control period. No adversesymptoms of withdrawal were felt after these two alleviating doses.(Note: in 4 day test subjects S1-S3 had experienced slight headache in24 hours after stopping the twice a day administration.)

Example 2.3 Clinical In Vivo Follow Up Test with Additional GeneExpression Analyses, Glucose Tolerance Test, and Plasma MetaboliteConcentration Analyses

In earlier in vivo experiments (Example 2.1 and 2.2) the last DGAcs wastaken in previous night. In this Example the last dose was double insize(=some 10 mg/kg), and it was taken in the same morning as thecollection of blood samples. Higher than “normal” therapeutic dose andshort time to blood measurement was chosen in order to see a clearerdose response compared to zero control. On top of very wide blood panel,like in Examples 2.1 and 2.2, also gene expression analyses fromperipheral leukocytes (Example 2.3.2 below), and concentration analysesfrom collected plasma samples (Example 2.3.3) was conducted. Alsoglucose tolerance test and related insulin measurements (Example 2.3.4below) was conducted. By comparing results of subject 1 (S1) fromExample 2.1 and in Example 2.3.1 (below), it is fairly straightforwardto see that this “high dose and immediate response strategy” wasefficient, and it produced even very important deviations in someparameters due to different administrations.

Example 2.3.1 Wide Blood Panel and Blood Count in Acute Test

In this Example the 4 day experiment in Example 2.1 was extended into4.5 day experiment by additional DGAcs dose in the morning before bloodmeasurement. Same wide fasting blood panel consisting of 25 differentmetrics and full blood count was measured for all participants (sampleanalytics by United Medix Laboratories, Finland).

Two healthy volunteers (S1 and S9) and two (otherwise healthy)volunteers using statin medication (S10 and S11) were chosen for theexperiment. The test set up was double blinded. S1 had participated inthe earlier 4 day experiment (see Table 3 above and also to a 3 weekpilot testing) and served as an important positive control on theefficacy of the DGAcs. Statin group on the other hand was chosen as somekind of a negative control because statins to some extend can possiblycounter act the positive effects of the DGAcs and on the other hand alsohave similar health effects. Statins suppress mevalonate pathway byinhibiting HMG Coa reductase activity and also disturb balancing ofcellular NADPH/NADP⁺ levels (FIGS. 3b and 4). On the positive side,statins have been shown to increase expression of inducible hemeoxygenase (HO-1) like the DGAcs can also do, and also to increaseLDL-receptor synthesis like the DGAcs can also do through Nrf2 pathwayactivation. Statin treatment group was not expected to yield positiveresults but possibly vice versa. For S10 the statin dose was halved twoweeks before the test from 20 mg to 10 mg (Simvastatin). For S11 statintreatment was kept at 20 mg per day (also Simvastatin).

TABLE 5 4.5 day human trial with 5 mg/kg/twice a day, and last dose 10mg/kg in the morning. DGAcs was mixed to water in advance. Two healthymale subjects with good physical condition and lean, age 47 and 50. (N =2) Measurements 2.5 hours after last dose Standard fasting blood test.S1 S1 S9 (4 (4.5 (4.5 day) day) day) S-Afos −2% 8% 12% Ratio AST/ALTdeclines S-alat −14% 18% −36% significantly for most of the S-Alb −2% 5%10% tested. S-Amyl 65% −5% −3% Important deviation from S-Asat −33% −14%−43% earlier. S-Bil −19% 4% 41% Important deviation from S-Bil-Kj −17%7% 40% earlier. S-Cal −2% 4% 3% Deviation in S-Cal prob. due to S-CK−22% −14% −36% admin. fS-Fe −28% −2% −20% Ck down for basically allfS-Gluk 2% −2% 4% tested. S-GT 11% 20% −9% Fe down despite bilirubin S-K−2% −2% −13% and HO-1 up. fS-Kol −2% 4% −8% Increase is a deviation fromHDL −5% 1% −1% general pattern. LDL 2% 5% −9% Indicates decreased celldeath. fS-Krea −4% 3% 9% Blood urea declines for 8 out S-LDH −4% −4% −7%of 9 subjects. S-Mg −2% 5% 0% S-Na −1% 1% 1% fS-Pi 18% 13% −15%fS-Transf 10% 5% 8% fS- −32% −6% −23% Transferr.satur. fS-Trigly −5% −3%−5% S-Uraat −5% 4% −1% fS-Urea −7% −8% −39%

Similarities compared to 4 day non-acute dose test: In Table 5 the percent changes compared to 0—control from 4.5 day administration arepresented for healthy volunteers S1 and S9. As a comparison also theresults from 4 day treatment for S1 are presented in the first column.For S1 all the values in all zero control measurements and inmeasurements after DGAcs treatment were in the recommended rangesindicating good general health. Nevertheless there were again some clearindications towards even better general health: 1) AST/ALT (ratio)declined from 1.32 to 0.96 (earlier in 4 day test the decline was 1.36to 1.05) indicating improved function of the liver, 2) creatine kinase(S-CK) declined 14% (−22% earlier) indicating improved heart and musclefunction, 3) blood LDH declined 4% (−4%), and 4) finally blood ureadecline 7% (−8%) indicating improved renal function.

Deviations compared to 4 day non-acute dose test: There are also someimportant deviations in S1 results compared to the earlier results fromdifferent administration before the blood sample. Importantly thesedeviations, in this high dose experiment, were to the same direction foralso S9 (in table 5), S10 (data not shown) and S11 (data not shown).Deviations from 4 day test: 1) bilirubin and bilirubin conjugate wereboth up this time indicating increased HO-1 activity, 2) calcium levelin the blood (S-Cal) was now up slightly compared to clear tendency todecline in earlier studies, which is very likely due to relatively highdose of calcium salt containing 15% of calcium, and finally 3) bloodcreatine was now up for all 4 subjects tested, earlier creatine(fS-Krea) levels tended to decline for all tested (see table 3).

For statin group (S10 and S11) there was very little internal orexternal consistency (except for above mentioned measurements), whichwas rather expected. The DGAcs does not work well, at least in shortterm and in high dosing, with statins because some natural pathways areinhibited; secondly because in high dosing HO-1 expression is clearlyactivated by the DGAcs. Statins have been reported to elevate HO-1expression as well, and thus even the zero controls of the statin groupalready contain this important therapeutic element from high dosing ofthe DGAcs, which makes it more difficult to reveal any differencesbetween the treatment and the control groups.

In statin group nevertheless, increased level of blood triglycerides(+83.5% for S10 and +25.6% for S11) is an additional element in linewith results of S2, S3 and S4 in Table 3. Increased blood triglycerides(TGA) likely indicate increased synthesis of TGA by the liver and theirtransportation for use in beta oxidation for other tissues e.g. musclesand heart (see also Example 3). For S1 and S9, with relatively highaerobic capacity and good physical conditions, it is hypothesized thatmuscle cells have developed their own glyceroneogenesis and fatty acidsynthesizing capacity, and thus the need to transport them from theliver is reduced.

It can be deduced from above clinical examples 2.2 and 2.3.1: The DGAcscan reduce oxidative stress of the cardiovascular system also in thelong term low dose administration. This reduction is likely due to dailystimulation of Nrf2/ARE systems as well as several other factors likereduction in blood pressure (FIG. 12). In higher dose and immediateresponse test the internal oxidative defense mechanisms, like increaseof bilirubin/HO-1, are acutely activated indicating some oxidativestress. After analyzing the gene expression results in the next Example2.3.2, we will give the therapeutic interpretation on these seeminglyconflicting results from the use of the DGAcs.

Example 2.3.2 Gene Expression from In Vivo Blood Samples

In the above 4.5 day experiment samples of peripheral leukocytes werecollected from S1 and S9 in fasting condition (0 h), and also 1 hour (1h) after taking 75 grams of glucose (Glutole, Biofile Pharma, 330 ml)for glucose tolerance test. Additional gene expression measurement wasdone from a separate 12 hour treatment for S1 and S9. In the 12 h testonly two last doses of DGAcs were administered, first dose 12 h earlierthan the blood sample, and the last/second dose 2.5 h earlier in thesame morning (like in 4.5 day experiment). For the statin group, S10 andS11, only one gene expression measurement was done in two hours afterthe fasting blood sample. Leukocytes were immediately separated fromblood samples, and after separation immediately lysed by stop solutionand stored in freezer according to instructions by the service provider.

Use of peripheral leukocytes as biomarkers of diseases: it is generallyknown that peripheral leukocytes and platelets can act as biomarkers ofmitochondrial dysfunction associated with several diseases includingdiabetes, neurodegenerative diseases, atherosclerosis and cancer. Forexample, in a study of mononuclear cells in type2 diabetes showed thatthe mitochondrial mass was decreased and that the mitochondria werehyperpolarized. Mitochondrial complex I activity was found to bedecreased in aged platelets and those obtained from patients withAlzheimer's disease had higher mitochondrial membrane potential thancontrols. Furthermore, platelets derived from normal individuals with amaternal history of Alzheimer's had lower cytochrome c oxidase(=MT-CO1.see below) activity.

For S1 and S9 there were altogether 4 measurement points for geneexpression, and only 3 repeats for each measurement. Results arepresented in Table 6 below; changes of gene expressions compared to thecontrol from peripheral leukocytes are presented. The Gene Expressionanalyses was conducted using TracTechnology by PlexPress Ltd inHelsinki. The TRAC data presented in Table 6 has been generatedaccording to process instructions with quality tested instruments andreagents. PlexPress Quality Management System is set up according to theISO 9001 standard. P-values are from t-distribution of a one sided testof deviation from control average, and are presented only when below10%.

TABLE 6 Results from gene expression study for S1 and S9 (combined data)S1 and S9, Combined Data 12 hour + 0 h test 12 hour + 1 h test 4.5 day +0 h test 4.5 day test 1 h test Gene Change % P-value Change % P-valueChange % P-value Change % P-value HO-1 −31% 4.0% −54% 4.7% 82% 0.1% 110%0.4% CYP1A2 100% 5.0% n.m. 62% 0.3% 14% CYP3A4 −17% −45% 67% 1.1% 69%CYP2C9 8% 18% 0% 46% CYP2B6 −14% 8% 73% 0.3% 147% 0.1% PGC-1a −3% −21%102% 0.4% 30% MT-CO1 −10% 3.3% 4% 24% 0.6% 23%   1% GPD2 24% 1.4% 23%3.2% 31% 0.2% 0% MT-CYB −2% 2% 22% 0.5% 21%   1% G6PD 3% 18% 0.1% 9%4.1% −13% GRHPR −26% 0.5% 6% 70% 0.4% 100% 0.1% AOX1 −6% 9.4% 8% 3.2%n.a. n.a. n.a. n.a. n.m. = data missing or not meaningful, n.a. =measurements not available (not conducted), P-values < 0.10 arepresented in the table

CYP1A2 is statistically significantly up regulated in 12 h and alsoafter 4.5 days before glucose intake, i.e. in fasting diet compared tocontrol. CYP3A4 and CYP2B6 are clearly up regulated after 4.5 days infasting condition and also after glucose intake. Inducers of the lattergenes/enzymes are used as anticonvulsants and mood stabilizers, thus theCYP3A4 and CYP2B6 data indicates potential therapeutic effect for theDGAcs (see indication areas). CYP3A4 was statistically significantly upregulated also for S10. Statistically significantly up regulated CYPsare located in the ER indicating increased ER activity from the use ofthe DGAcs.

Next important observation from Table 6 is the sharp increase in HO-1expression after 4.5 day administration. The increases in both fastingcondition (0 h) and 1 hour after glucose intake (1h) were bothstatistically very significant. Furthermore Similar statistically verysignificant 160% increase in HO-1 was observed also for S10 in statingroup. (S10 was the test subject with halved dose of Simvastatin.)Earlier presented results in Table 5 of increased blood bilirubin levelsconfirm that the increased HO-1 gene expression has lead also toincreased enzyme activity. (This is just the opposite as in Table 3administration.)

Importantly HO-1 gene expression was clearly down regulated in the shorttest of 12 hours/2 doses of DGAcs. This same phenomenon happened in 4day test earlier, as indicated clearly by the decrease in bloodbilirubin levels in Table 3 (and also in Table 4).

Interpretation: One can conclude that HO-1 expression is not highlyactivated immediately after starting the use of the DGAcs or in factthat its expression was even down regulated. The same down regulationseems to be the case when there is longer time from the administrationeven in high dose case Table 3) and also in the case of lower once a daydose (Table 4). At the same time immediate dose response for DGAcs canclearly increase HO-1 expression very significantly. Interpretation ofthese apparently but not in reality conflicting results: HO-1 isactivated by the use of DGAcs de facto always but only temporarily.Oxidative stress induced by the increase in aerobic ATP production(OXPHOS) can also be efficiently ameliorated due to up regulation ofNrf2/ARE genes; both are due to the use of DGAcs (see FIG. 2).

Therapeutic strategy for the DGAcs administration with HO-1/Nrf2/AREgenes: extremely many diseases (see therapeutic areas) can beameliorated by controlled up and down regulation of HO-1 and otherNrf2/ARE genes. With right dosing the DGAcs can be used for diseasesthat need e.g. activated inducible heme oxygenase (HO-1) system or otherNrf2/ARE enzymes. The DGAcs can work like a vaccine that activates(sometime permanently) the immune system against certain diseases, i.e.the use of the DGAcs can keep HO-1 and other Nrf2/ARE genes in activestate by daily temporary stimulation. In case of a successful mild dailystimulation, Nrf2/ARE genes are down regulated for most of the time andalso ROS levels are lowered. For some disease states high dosestemporarily might be needed in order to stop harmful (e.g. inflammatory)processes by strong activation of Nrf2/ARE response. Simultaneousincrease in aerobic energy metabolism and pyruvate production enhancetherapeutic effect significantly.

G6PD and AOX-1 (aldehyde oxidase) genes belong also to Nrf2 genes thatoften use NADPH-5enzymes as co-substrates in reactions (see FIG. 4).NADPH is also the favorite co-substrate for GRHPR gene and thus itprobably can be classified as an Nrf2 gene. Thus we could conclude that3-4 Nrf2 genes are up regulated by the DGAcs. (Unfortunately AOX1 wasnot yet included in the 4.5 day panel for S1 and S9. In the case of S10in 4.5 day panel that was conducted later, AOX1 gene expression wasincreased by 56% with P-value=0.1%.)

Important additional support to the notion that the DGAcs can stimulateendogenous antioxidant defenses (Nrf2/ARE) comes from consistently andstatistically significantly declining ROS levels in human hepatocytesexperiments (see Examples 1.1-1.6).

Interestingly GRHPR enzyme is first slightly down regulated evenstatistically significantly and up regulated strongly only after 4.5 dayadministration. Both are consistent observations. For the use of DGA theclear activation of GRHPR and its repeatable DGA-HPA loop and dimensiontowards peroxisomes is important.

Genes related to enhanced aerobic energy metabolism: GPD2, MT-CO1(NRF1), and MT-CYB relate directly to electron transport chain thatderives energy from NADH molecules (see definitions from FIG. 3b ).PGC-1a is a SIRT1 dependent master regulator of energy metabolism, e.g.of mitochondria biogenesis, and related multiple tasks. (PGC-1a isactivated or deactivated also via multiple other ways than NAD+dependent deacetylation by SIRT1.)

As can be seen from Table 6 MT-CO1 and MT-CYB are clearly andstatistically very significantly up regulated after 4.5 day use of theDGAcs. Importantly increased coding of MT-CO1, mitochondrially encodedcytochrome c oxidase I, can be directly link to NRF1, which is nuclearrespiratory factor 1. NRF1 functions e.g. as a transcription factor,that activates the expression of some key metabolic genes regulatingcellular growth and nuclear genes required for mitochondrialrespiration. Also PGC-1a is up regulated by over 100% and statisticallysignificantly in 4.5 day in fasting conditions. Its expression isincreased by 30% also 1 h after glucose intake, but due to poor dataquality in some observations this increase is not statisticallysignificant. Furthermore mitochondrial glycerol phosphate dehydrogenase(GPD2) is up regulated already in 12 h and continues to be that also infasting conditions after 4.5 day administration.

Gene expression data points very clearly to the direction that the DGAcscan activate aerobic metabolism and mitochondrial biogenesis. Many ofthe up regulated genes (also CYPs) encode heme proteins containing iron(Fe). This is nicely in line with decrease of free Fe in blood sample(see Examples 2.1, 2.2 and 2.3.1). Additionally in rat experimentsmitochondrial up regulation by increased calcium release was observed(Example 2.2). The data as a whole, point clearly to the direction thatthe DGAcs can reduce mitochondrial dysfunction by activating biogenesisof new properly functioning mitochondria. Thus we postulate that theDGAcs can prevent diabetes, neurodegenerative diseases, atherosclerosisand cancer by decreasing mitochondrial dysfunction. One example:according to literature decreased MT-CO1 activity in leukocytes has beenassociated to increased risk in developing Alzheimer's disease.

Immediate response of the use of the DGAcs seems to be the activation ofmitochondrial glycerol phosphate shuttle gene (GPD2). This phenomenonmay be useful in fast exercise performance and recovery from exercise(Example 6). GPD2 can temporarily generate ATP energy much faster fromNADH than traditional Malate-Aspartate-shuttle. Also acidosis andlactate production is decreased.

Example 2.3.3 Plasma Sample Analyses on PYR, ALA, LAC and NO

As mentioned above also plasma samples were collected from S1 and S9, at0 h and 1 h both in 12 h and 4.5 day test, i.e. 16 observations weregathered with either DGAcs or zero control situations. Additionally instatin group (S10 and S11) 2×2 measurements were done. Altogether therewere 20 observations and, thus 10 differences between control and DGAcsadministration can be calculated. Pyruvate concentrations were measuredusing Pyruvate Assay Kit from Sigma (MAK071 Sigma). Alanineconcentrations were measured using Sigma Alanine Assay Kit (MAK001).Lactate concentrations were measured using Sigma Lactate Assay Kit(MAK064). And finally nitric oxide (NO) concentrations were measuredusing OxiSelect™ In Vitro Nitric Oxide (Nitrite/Nitrate) Assay Kit fromNordic Biosite Oy.

TABLE 7.1 Deviation in Plasma Pyruvate, Alanine and LactateConcentrations from Zero Control Correlation between Change the levelsof plasma Meas- Change in in Change in PYR & PYR & urement PyruvateAlanine Lactate ALA LAC  1 13% 1% −30%  2 59% 5% −63%  3 23% 4% n.a.  439% 21%  −48%  5 35% 33%  −75%  6 −22%  −8%  −28%  7 32% −21%  −70%  832% 3%  46%  9 (S11) 11% 3%  −1% 10 (S10) 38% −6%  −18% Average 25.9%  3.5%   −31.9%   Change of the Con- centration P-value  0.22%**) 24.0%    1.8%*) +0.827**) −0.794**) compared to the control *)P-value <0.05,**)P-value <0.01

As can be seen from Table 7.1 plasma pyruvate amount increases onaverage by 25.9% compared to control measurement. This increase happenedin all tested, i.e. also in statin group, and is statistically verysignificant. In healthy volunteer group only, i.e. S1 and S9, theincrease was also approximately 25%, but more importantly it remainedroughly at 20% also 1 hour after administering 75 000 mg of glucose. TheDGAcs administration increases intracellular pyruvate production both infasting conditions and also after glucose administration. This indicatesthat the pyruvate comes from multiple sources like presented in FIGS. 1b, 3 a and 3 b. Lactate seems to be clearly one very natural source seenfrom the results.

Plasma lactate amount decreases on average by 31.9% compared to controlmeasurement. This increase happened in all tested, i.e. also in statingroup, and is statistically very significant. In healthy volunteer grouponly, i.e. S1 and S9, the decrease was even bigger some 38%. As expectedthe correlation between the levels of plasma pyruvate and lactate wasnegative and roughly −80%. In the plasma there is roughly ten times moreof lactate than pyruvate. Almost perfectly in line with that themultiple in regression analyses was −012 when the level of observedpyruvate in plasma was explained by observed level of lactate.Furthermore these results seem to be also very well in line with socalled lactate cycle in which lactate is transported to the liver infasting state from gluconeogenesis. In fasting situation the lactatetransport was on average 84% higher compared to situation after 75 000mg glucose intake, and the difference was statistically significant(P=1.1%).

Extra cellular pyruvate is extremely good source of aerobic energy foractive cells and tissues containing multiple mitochondria, e.g. neurons,inner organs, and aerobic muscle cells. Pyruvate is also a very suitableintracellular (starting) building block in anabolic reactions, see FIG.3a . Plasma Alanine levels did not change compared to control. This isalso interesting because normally alanine increases as a consequence ofincreased pyruvate. Possibly peroxisomal transaminase reactions from HPAand ALA into PYR and L-Serine (FIG. 1b ) counteract the increase in ALA(and aKG) from PYR (and glutamate).

Decrease in lactate cycle is very good because it e.g. is a clearindication that cells are able to use bigger part of the nutrition thatthey are provided themselves. Very significant decrease in blood lactatecompared to control is also a clear indication that mitochondrial NAD⁺providing shuttles work better in the DGAcs group. We also see this verystrong result as a sign that likely RBCs have increased their lactateintake somewhat. Ntf2/ARE mechanism and DGA-HPA—loop can work also inRBCs, even without mitochondria, and thus the DGAcs can enhancemetabolism even in the RBCs.

There is also a surprising additional follow up on increased bloodpyruvate concentrations: a decline in blood urea (like seen in Examples2.1 and 2.3.1). An increase in pyruvate increases the flux of TCA e.g.in renal cortex mitochondria. In literature it has been shown and it isalso theoretically obvious that pyruvate increase in turn increases aKGconcentration in the matrix and decreases intramitochondrialNAD⁺/NADH-ratio. Both processes decrease the activity of glutamatedehydrogenase (GLDH) activity. As is well known, GLDH reaction providesmajority of amino groups (NH₃) into the urea cycle. I.e. on top ofactivation of mitochondria and aerobic metabolism, the use of DGAcs canalso save amino groups for protein synthesis e.g. in muscles. Additionalnote: pyruvate increases and intramitochondrial NAD⁺/NADH-ratio declinesalso due to increased beta oxidation induced by the DGAcs. The follow upis the same but causes slightly different. In both cases ATP productioncapacity for e.g. anabolic reactions is enhanced.

Related to the decrease in urea cycle the NO level decrease amonghealthy volunteers whose BMI<25 (see Table 7.2). In the literature thisphenomenon is well known. Decrease in urea cycle decreases amount ofL-arginine. NO is synthesized from the reaction from L-arginine tocirtulline.

TABLE 7.2 Deviation in Plasma Nitric Oxide Concentrations from ZeroControl Change in Change in Nitric Oxide Measurement Nitric Oxide (BMI<25)  1  −8%  −8%  2  −9%  −9%  3 −32% −32%  4 −25% −25%  5 −10% −10%  6 −1%  −1%  7 −19% −19%  8  +8%  +8%  9 (S11)  +22%*) — 10 (S10) −25%−25% Average −9.9%  −13.4%   Changes in NO are in Change of the linewith seen decline Concentration in urea cycle. P-value   4.25%*)  0.63%**) Possible clear compared to deviation in BMI >>25 the controlgroup in NO vs. urea can be observed but needs more studies. *)P-value<0.05, **)P-value <0.01

It is very interesting and also consistent to notice that observeddecline in NO (approximately 10%) is roughly the same as average declinein blood urea after 4 and 4.5 day administrations (approximately 9%).

Even more interesting is the observation that in S11, belonging to highstatin group and BMI >>25 (measurement 9), NO production was increased.All plasma samples were divided into 4 wells to get more accurateaverage readings. We can use these 4 independent measurements (from thesame sample) to test that is the deviation in NO of S11 statisticallysignificant. The increase of NO from control turns out to bestatistically significant (P=1.4%).

Example 2.3.4 Glucose Tolerance Test, Plasma Glucose and InsulinCompared to Zero Control

In the 4.5 day experiment also standard, fasting glucose tolerance testwas conducted for S1 and S9 starting after 2.5 hours of lastadministration of DGAcs. Blood glucose levels were measured at 0 h, 60min and 120 minutes.

In all 120 minutes measurements (with S1 and S9, altogether 4comparisons) blood glucose was lower compared 0 h level in DGAcs groups.For S9, who had slightly elevated fasting blood glucose level of 6.0mmol/L the drop in glucose level was more significant, i.e. some 0.6mmol/L lower at 120 min compared to the control. For S1 the drop wasseen but marginal, probably due to relatively low starting level of 5.0mmol/L. Blood insulin levels were measured at 60 min. For S9 higherinsulin level with the DGAcs was seen at 60 minutes, and that likelylead blood glucose rapidly down from elevated levels. For S1 bloodglucose levels did not rise significantly at 60 minutes, and thusinsulin levels at 60 minutes did not show any pattern.

All in all the DGAcs seems to have a positive decreasing effect on bloodglucose level after 75 grams glucose intake. This is likely mostly dueto increased ATP production capacity of the cells that facilitatesglucose intake and conversion into glycogen, and G6P to be used inpentose phosphate pathway and glycolysis (see FIG. 3b ). Also insulinproduction by pancreatic beta cells (that uses e.g. GP-shuttles in theirenergy production) probably rise, and that also enhances the processtowards faster glucose homeostasis in blood stream.

Example 3

NMDA-Induced Excitotoxity in Rat Cortical Neurons

Two separate studies were conducted. First study conducted withUniversity CRO and second confirmatory study with Private CRO.

Cell culture. Cortical neurons were dissected from P1 rats and culturedin 96-well plates up to 7 days in the medium containing: neurobasalmedium, 1.5% B27 supplement, 1 mM L-glutamine, penicillin/streptomycin.

Treatment of cells with DGAcs was done in two different ways. In allstudies the total cultivation time was 7 days (7^(th) day invitro=7DIV). In the first study a clear element of calorie restriction(CR) was built in to the standard model provided by the CRO. The other24 hour pretreatment was without CR (more info in examples 2.1 and 2.2below). Separately also exceptionally high doses of DGAcs wereadministered to neurons. No toxicity was observed even with 40-200 timesthe effective treatment dose of DGAcs. (Effective dose was 10-50 μg/m).

Cell viability/cytotoxicity. Cell viability was detected by LDH testslike in examples 1.1-1.2.

Fluorescent imaging. Imaging experiments were performed at 7DIV. Thecells were loaded with fluorescent dyes (fura4F, fura-ff and rhodamine123,) in cell culture medium at 37° C. 1 h, 5 μM for each dye. Thencells were washed with Mg²⁺-free Locke's buffer containing 1.3 mM Ca²⁺,moved to the microscope stage, and imaging was performed at roomtemperature. For NMDA-induced Ca²⁺-peak measurement a higher affinityCa²⁺-indicator Fura4F (Kd 0.77 μM) was used. For delayed calciumderegulation experiments a lower affinity Ca²⁺-indicator FuraFF (Kd 5.5μM) was used to avoid a saturation of the signal.

Example 3.1 Protection Against NMDA-Induced Excitotoxity

Cell treatment: concentration of DGAcs (10, 50 and 100 μg/ml) was addedinto cell culture medium at 4DIV. After 24 h (at SDIV) 25% of medium waschanged to fresh containing 1.5x concentration of DGAcs. Same treatment(change 25% with 1.5× concentration) was repeated at 6DIV. The cellswere transferred into Mg²⁺-free Locke's buffer containing the sameconcentrations of DGAcs immediately before imaging experiments (at7DIV). For controls the same amount of medium was changed withoutaddition of DGAcs.

Measurement of LDH Viability, Effect of Calorie Restriction (CR)

Cell treatment according to the protocol renewed only 25% of the mediumduring SDIV and 6DIV, meaning that neurons received only very smallamounts of new nutrition, i.e. experienced calorie restrictions (CR).Again CR caused dose dependent viability loss DGAcs groups, this time inneurons (see FIG. 13). This same phenomenon of increased cell death inCR was already seen with human hepatocytes in previous examples. Likelyexplanation is the same: DGAcs administration increases metabolicactivity in neurons and/or their (aerobic) ATP production andconsumption, which leads to enhanced cell cycle control and programmedcell death (apoptosis) in nutritional scarcity. As can be seen from FIG.13, this effect is very small but on the other hand consistently dosedependent and statistically significant. Viability loss compared to zerocontrol was highest and statistically significant in 10 μg/ml group butalso all other groups experienced loss of viability.

Measurement of LDH Viability, Protection Against Excitotoxity Induced byNMDA Stimulation

In analyzing the results from the test set up starting at 7DIV with 1 hNMDA stimulation (and 23 h follow up period), separate viability losscaused by CR can be taken into account by indexing 25 μM NMDA and 50 μMNMDA groups with the results of 0 NMDA control (see FIG. 14a ). As canbe seen from the figure, DGAcs treatment induces very clear andsignificant protection against NMDA-induced excitotoxity in both 25 μMNMDA and 50 μM NMDA group. Even without correction for CR inducedviability loss, DGAcs treatment induces very clear protection againstNMDA-induced excitotoxity in 50 μM NMDA group (see FIG. 14b : “Viabilityafter 24 hours with 1 h NMDA stimulation, indexed to 0 NMDA”)

In literature it is described that excitotoxicity may be involved inspinal cord injury, stroke, traumatic brain injury, hearing loss(through noise overexposure or ototoxicity) and in neurodegenerativediseases of the central nervous system (CNS) such as multiple sclerosis,Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson'sdisease, and also Huntington's disease. The use of DGA can effectivelyprotect neurons against excitotoxic injury and thus likely also toprevent the onset of and/or alleviate mentioned diseases related to it.

Study set up in example 2.1 with primary rat cortical neurons wasrepeated with Private CRO in two similar experiments. In the firstconfirmatory study the viability loss due to CR was confirmedstatistically significantly at 6DIV. This demonstrates that DGAcs causesloss of viability when calories are restricted in vitro, and this effectmust be taken into account in the model. As already explained above,observed small loss of viability is likely due to two factors:1)increased aerobic ATP production (PGC-1a/NRF1) and related increase inenergy and nutrition consuming anabolic, and anaplerotic reactions, and2) enhanced cell cycle control (e.g. Nrf2/ARE).

Despite the success in repeating energy metabolic part of thisexperiment with neurons, the NMDA protection part was not as successful.Results from excitotoxity repetition by the private CRO were in linewith above results but they were not statistically significant. Theinstability of the results may be due to the fact that e.g. Nrf2/AREactivating mechanisms are mostly missing from neurons. Nrf2-mediatedneuroprotection is primarily conferred by astrocytes both in vitro andin vivo. Neuronal cultures would need astrocytes and other glial cellsfor full protection by the use of DGA. i.e. important Nrf2 activation bythe use of DGA cannot be at full play in neuronal cell culture. In pureneuronal cell culture model provided by the CROs with minimum amount ofglial cells only the enhancement of mitochondrial energy metabolism bythe use of DGA can functions at full strength. Worth noticing is thatthe increase in energy metabolism gives also alone protection againstexcitotoxic insult like indicated also by the next Example 2.2.

Example 3.2 Mitochondrial Ca²⁺-Uptake in Rat Cortical Neurons

In this study azero control and one DGAcs group of primary rat corticalneurons was cultivated normally without DGAcs until 6DIV, and then theDGAcs group was treated with 50 ug/ml DGAcs for 24 hours. After thatculture medium was replaced by Mg²⁺-free Locke's buffer containing 50ug/ml DGAcs and imaging experiments were performed at room temperatureat 7DIV.

50 μM NMDA in the presence of 10μM glycin in Mg²⁺-free buffer was usedto stimulate the cells. For NMDA-induced Ca²⁺-peak a higher affinityCa²⁺ indicator Fura4F (Kd=0.77 μM) was used.

For complete mitochondrial depolarization and release of Ca²⁺accumulated in mitochondria 2 μM protonophore FCCP was used in thepresence of NMDA-receptors inhibitor MK801 (2 μM). Results of calciumrelease for zero control and DGAcs group in various treatments arepresented in Table 8.

TABLE 8 Results from Study testing Calcium uptake by Mitochondria ofPrimary Rat Cortical Neurons baseline Ca peak NMDA FCCP Zero ControlAverage Ca release 0.073 0.213 0.402 N 5 5 5 Standard error of the0.00198 0.0127 0.0295 mean (SEM) 50 ug/ml DGAcs for 24 h: Average Carelease 0.070 0.219 0.475*) N 3 3 3 Standard error of the 0.003460.00395 0.00667 mean (SEM) *)Statistically significant deviation fromthe control (p = 3.4%)

As seen from complete mitochondrial depolarization treatment (FCCP) intable 8, calcium uptake by the mitochondria is increased by 24 hour ofDGAcs administration. It indicates that the activity of mitochondriaand/or their mass has increased, e.g. due to mitochondrial biogenesis.This result is in line with similar activity increase in gene expressionexperienced in peripheral leukocytes after DGAcs treatment in humans invivo (see Example 2.3.2).

Clear enhancement of energy metabolism of CNS tissues is also in linewith indirect results from clinical in vivo experiments, e.g. head acheand other withdrawal symptoms after stopping administration (see Example2.1 and 2.2).

Example 4 Weight Loss and Change in the Body Composition, a 3 Week InVivo Study on Rats

In this in vivo experiment 45 rats (24 males and 21 females) were testedin Finnish National Institute of Health. Used DGAcs doses were highcompared to human trials ranging between 100-1000 mg/kg/day, but on theother hand the metabolism of rats is much faster than in humans and thusthe effective doses for rats can be clearly higher than for humans. Insome experiments even 20-50 times higher compared to humans in vivo havebeen reported in literature. Thus this experiment can give indicationalso for human use. The animals were kept under controlled conditions,with the temperature set from 20° C. to 21° C., the humidity at 47.6%,and a 12-hour light-dark cycle (lights on at 6:00 am). In this adlibitum feeding study the DGAcs was mixed in the diet for the wholeexperimental period (3 weeks). The animals were divided into 4 groups,which received 0 (zero control), 2, 10, and 20 g glycerate per kilogramof diet. The average DGAcs consumption was 0.1, 0.5, and 1.0 g/kg perday, respectively, as calculated based on weekly food consumption. Theanimals experimented were 3 to 5 months old AA rats. The animals weregiven water and a standard laboratory feed (SDS RM1) ad libitum for 3weeks. In the experiment the development of weights of rats receivingDGAcs, and the control were measured, as well as the food consumptionduring the experiment and change in the body weight. Males and femaleswere separated resulting altogether to 8 groups with 5-6 animals ineach.

In male groups combined, the average daily food intake was roughly 50g/kg/day, i.e. 5% of the total body weight (bwt). In female groups itwas slightly higher some 56 g/kg/day. As a cumulative sum the rats ateroughly their own bwt of feed during the 21 day test period.

Both in male and female groups the food intake was higher in DGAcsgroups compared to the control, 3.7% in males and 2.5% in females.Interestingly in male DGAcs groups combined this increase in foodconsumption was even statistically significantly different from thecontrol group. This is very remarkable because at the same time theaverage body weight in male rats decreased 4 grams or 1.1% of bwtcompared to the control. Further to stress the point, also in femalesfood intake was elevated by 2.5% and the average weight remainedpractically stable, increasing only 0.6 grams or +0.2% compared tocontrol. (In female DGAcs groups there was clearly more deviations inthe first week after starting the test, and that is one reason why theincrease in food intake was not statistically significant.)

Otherwise everything in this experiment has been equal between differentgroups. Daily energy consumption into physical activities was similar ingroups and there were no running wheels or any other equipment thatcould cause significant differences in the amount of daily exercise. Thetemperatures were kept stable and at neutral levels, and circadianrhythm was stable. Thus it is unlikely that differences in exercise orthermogenesis could explain observed clear increase in food consumptionand simultaneous average drop in bwt in DGAcs groups.

Moderate, but statistically significant, 2.5-4% increase in food intakeparadoxically leads to a decrease in weight (or stable weight in thecase of female rats) in DGAcs groups. Energy consumption has to beincreased by the use of DGA. This phenomenon was observed already incell culture experiment with human hepatocytes and rat cortical neurons.Now this odd phenomenon of increased energy consumption has beendemonstrated also in vivo in rats. The amount of unexplained energyconsumption is roughly 5% in males. Furthermore in section “ATPproduction per gram of nutrition and change in body composition” weconcluded that, ATP energy production per gram of nutrition increases bythe use of DGA. This means that unexplained energy consumption is evenwider in this in vivo experiment, maybe even 5-9%.

The use of produced extra ATP energy is very likely to anabolicreactions, e.g. gluco-/glyceroneogenesis and protein synthesis thatconsume a lot of energy (see FIGS. 3a and 3b , and starving diet and CRin example 1 and Example 3.1). Energy is consumed also to increasedcontrol and correction of anabolic processes in the ER, e.g.ATP-dependent chaperones. Increased supply of substrates, pyruvate andamino groups, for anabolic reactions by the use of DGA (shown in example2.1-2.3) supports also the idea that excess ATP is consumed for enhancedrenovation of proteins (enzymes) and similar complex macromolecules. Ingeneral these processes increase ATP consumption and they are beneficialto cells and promote long term health.

From the above and Examples 5 and 2 follows the next important finding:by increasing ATP production and beta oxidation of fatty acids (FA), theuse of DGA can have an effect on body composition of humans and also onanimals, like livestock, poultry and fish. Energy stored in fats isindirectly converted into protein and thus increasing the muscle contentof the body in the expense of fat.

Example 5

Enhancement of Mitochondrial Beta Oxidation in Longer Term, In Vivo inRats

From the above experiment altogether 12 rats were chosen from 0.1g/kg/day DGAcs group (N=6) and zero control (N=6) for a measurement offree glycerol from liver samples. Liver samples contained also bloodentering the liver from other tissues, thus on top of hepatic tissuesthe samples reflect also the metabolic situation in other tissues of thebody than just the liver.

In this experiment nutrition intake, except of course for DGAcs, wascontrolled to be exactly the same during the experimental day, and threeday before the experiment. For AA rats, that are used to alcohol butdidn't receive any alcohol in 3 day before the experiment, catabolicredox -activity in the cytosol was controlled by giving rats ethanol(equaling 1.2 per mille blood alcohol concentration, which representsonly mild alcohol intoxication to the AA rats). The rate of ethanoloxidation remained the same in all groups. The maximum increase was onlysome 3.6% acceleration compared to control, i.e. in practice the same,and naturally this deviation was not statistically significant.

Experimental session started at 8:00 to 9:00 am. To diminish possiblepain during the experiments, all animals were injected subcutaneouslywith buprenorphine (0.01 mg/kg) a few minutes before the blood samples.During the experimental session no difference in other metabolicactivity, e.g. rate of ethanol oxidation, between the control andtreatment group was detected from blood samples. Immediately after thelast blood sample, the animals were anesthetized with pentobarbital (40mg/kg IP, 1% [wt/vol] in saline). Thereafter (5-10 minutes), liverpieces were quickly (within 2-4 seconds) excised and freeze-clamped.Liver samples were stored at -718 C until glycerol determination. Forthe liver free glycerol measurements, the freeze clamped livers werethoroughly homogenized and diluted 1:6 with mQ water, assuming the livertissue density of 1 g/mL. The homogenates were incubated in a boilingwater bath for 5 minutes and centrifuged at 14000 rpm for 15 minutes.The resulting supernatants were used for free glycerol measurement withan enzymatic end point commercial assay kit (Boehringer-Mannheim,R-Biopharm, Darmstadt, Germany) according to the manufacturer's protocolexcept that the assays were modified for use with small volumes ofsupernatant using a 96-well microplate reader (Labsystems Multiskan RS,Helsinki, Finland). Samples were assayed in duplicate.

TABLE 9 Effect of DGAcs on hepatic free glycerol levels, including alsoextracellular matrix Treatment Free glycerol concentration Control (noDGAcs) 3.06 +/− 0.55 (6) DGAcs (0.1 g/kg per day) 4.88 +/− 1.21 (6)* *)P< 0.05 compared with the control group Glycerol levels are expressed asmicromoles per gram wet weight tissue

In 0.1 g/kg DGAcs group glycerol levels were increased by 59% comparedto the control. This result is very remarkable. The observed increase inthe concentration of free glycerol compared to the control samples wasroughly 0.17 g/kg, i.e. even clearly bigger than the average daily dosein DGAcs group.

It should be noted that in this experiment there was no acuteadministration of DGA before the experiment, and there is no reason toexpect that DGA would accumulate in the liver during the 3 weekexperiment, because the liver is very efficient in all metabolisms(especially towards glycolysis and gluco-/glyceroneogenesis via GLYCTK1or GLYCTK2 enzymes, FIG. 1b ). Also the fact that ethanol oxidationdidn't change compared to the control clearly indicates that the amountof DGA in the liver was approximately the same as in control group atthe start of the experiment, i.e. very small in both.

Furthermore from other conducted experiments (see Example 2) we knowthat DGA seems to be evenly distributed into various tissues afteradministration in vivo. Also in all other tissues DGA should berelatively easily metabolized. Thus also in other tissues theconcentration of DGA, at the onset of this non-acute dose experiment hasbeen clearly less than 0.1 g/kg, maybe at maximum some 0.01 g/kg. Thismeans that direct or indirect acute conversion of DGA to free glycerolcannot explain the result in Table 9, not even any significant part ofthe result.

First and probably the main source of the difference in free glycerol inthis experiment is from triglyceride lipase activity that liberatesfatty acids from trigys for mitochondrial beta oxidation in the liver.In the liver it is also possible that free glycerol arises fromD-glyceraldehyde (D-GALD) with alcohol oxidation, but that happens inthe same amounts in both groups and thus cannot explain observeddifference in free glycerol. Additionally there can't be any tendencyfor building of large quantities of D-GALD molecules into the liver by 3week DGA treatment that could explain large difference in free glycerateobserved in this study. (Liver manages most of fructose metabolism inthe body and it produces D-GALD. The natural metabolic direction forD-GALD is towards glycolysis by triokinase enzyme (see FIG. 1b ).)

Supporting the increase in beta oxidation as a source of the hugedifference in free glycerol is the fact that in muscle tissues only theFAs of circulating trigys are taken in to the myocytes and the freeglycerol part is liberated to the blood circulation to be taken back tothe liver. Thus increased beta oxidation in muscle cells is also verymuch in line with glycerol increase in the liver sample in the DGAgroup.

Ruling out other possible sources for the difference in free glycerol:Glycerol kinase catalyzes the reaction from free glycerol+ATP toG-P-3+ADP (see FIG. 1b ). This enzyme works also to the other reactiondirection, but in practice ADP has been found very unappealing substratefor this kinase enzyme. Thus observed huge difference in free glycerolcannot be directly from increased G-3-P formation from glyceroneogenesisin DGA group. DGA does not easily convert towards D-GALD becausealdehyde dehydrogenase (ALDH) enzymes favor clearly the oppositedirection (FIG. 1b ) and in any case are ALDH5 are often located andactive mainly in the mitochondrial matrix. AOX1 enzyme (see Example 2.3)could be a possible enzyme for mentioned reaction (direction) but it isvery unlikely that it could facilitate the volumes needed forextraordinarily high 59% increase in free glycerol. (Furthermore inconducted one hepatocyte study the expression of AOX1 did not increaselike it did with leukocytes.) All in all the acute conversion towardsD-GALD and thereafter towards glycerol can represent only a very minorpart of the total even in “highest” cases. Thus DGAcs administrationdoes not directly cause the observed increase in glycerol via thatroute. Second favorite direction for DGA is towards HPA. According togene expression analyses this direction is activated at least in thelonger term (see Example 2.3). This direction does not provide glycerol.Finally very likely the long 3 week DGA administration with the food hasincreased the rate of glyceroneogenesis compared to the control(independently of an increase in aerobic metabolism). That of course canindirectly increase the amount of free glycerol in the hepatic tissuesthrough an increase in beta oxidation and required lipase reactions thatliberate free glycerol inside the liver and in other tissues (to betransported back to the liver). Increase in this pathway is in factsomething that we want to prove here.

Because the difference in free glycerol in this non-acute dose study isso huge 59%, it is very likely due to a more structural increase inmitochondrial beta oxidation. We postulate that 3 week administration ofthe DGA has increased the use of FAs as a source of energy and thusmitochondrial beta oxidation has increased in the liver and otheraerobic tissues. Increase in beta oxidation is supported also e.g. byincreases in blood trigys levels in clinical Examples 2.1, 2.2, and 2.3in DGA groups compared to zero controls.

We can now summarize that the use of the DGA increases metabolic use offats for mitochondrial beta oxidation. Fatty acid oxidation yields a lotof ATP energy for ATP consuming activities e.g. cell cycle control andprotein synthesis. In experiment 2 we have shown that amino acid removalfrom the body is decreased by the use of DGA.

In Example 2.3.3 we show that lactate cycling from the cells to theliver is also decreased. In Example 4 we noticed some 2-5% imbalance inunexplained energy supply vs. consumption. This imbalance grows evenhigher, even to some 5-9%, when we can now assume that ATP generationper gram of nutrition is likely enhanced by e.g. the increase in aerobicenergy production (beta oxidation and decreased lactate cycling) by theuse of the DGA.

Higher energy level without increase in body weight is the best possibleoutcome that any pharmaceutical and/or health promoting substance canyield. The extra energy produced (supply) is used in 1) proteinsyntheses and other anabolic and anaplerotic reactions, and 2) inenhanced metabolic and cell cycle control. Energy stored in fat is insmall but meaningful scale converted into muscle tissue. Total bodyweight can decrease without losing muscle mass.

Example 6

Effect on Physical Performance and Recovery

Acidosis produces lactate that is an indication of restrain to fast ATPproduction. As seen in Example 2.3.3 the use of DGAcs can reduce plasmalactate levels on average by 30 percent. This very strong result hasbeen obtained in non-exercise state but nevertheless it shows that theuse of DGAcs can prevent acidosis formation by reducing lactate amountin blood.

Two healthy male volunteers S1 and S3 from Example 2.1 participated in400 m running experiment with 4 day administration of DGAcs and withoutit. The experiments were done at 2-3 pm in the afternoon. In DGAexperiment additional dose of DGAcs (5 mg/kg) was taken before lunch at11 am, and lunch was eaten at 12 am. Both volunteers possessed goodphysical condition but were not trained in 400 meter running. The lengthof the 400 m exercise at full speed was too much especially for S1. ForS3 no significant differences between control and DGA times wereobserved. Nevertheless, in qualitative terms, both S1 and S3 were muchmore ready to resume other physical exercise with the DGA than withoutit. This difference was striking especially for S3 and can be as aresult in decreased acidosis.

Additionally based on results from practical other Examples 1-5 it isobvious, that DGA can enhance energy metabolism and energy productionthus contributing also to enhancing physical training, performance andrecovery from exercise.

TABLE 10 Non-exhaustive summary table of the results from first set ofclinical studies, i.e. with non-acute dose/Examples 2.1 and 2.2 No. TestResults Indication Link to other results 1 fS-Alat All study subjectshad fS-Alat levels in the Functioning of liver. Reduction of fS-Ala isan AST/ALT - ratio declines due recommended range. Tendency forindication of an improvement in liver functions. to use of DGAindicating improvement was observed, when values improved function ofthe were higher in the range. (S3 is an exception liver. due toextremely hard physical training session before 4 day treatment.) N = 8.2 fS-Asat On average 15-20% reduction in values. N = 6. Functioning ofpancreas, skeletal muscles and heart. See also AST/ALT - ratio (seeabove). Reduction is indication of an improvement. 3 Bilirubin Onaverage some 20% reduction in values. Lower bilirubin level is anindication of increased viability of See also result bilirubin N = 6.erythrocytes and/or lower degradation of conjugate and fS-Uraat, i.e.heme proteins. Antioxidant HO-1 enzyme activity is lowered systemicoxidative lowered indicating that the oxidative stress is stress. DGAuse can also reduced, i.e. in this lower dose regime HO-1 is onlyactivate HO-1 gene and temporarily activated and on average it is downincrease bilirubin production regulated. (see example 2.3), which is atherapy option for some acute diseases and conditions. 4 Bilirubin Onaverage 20% reduction in values. N = 6 Increased viability oferythrocytes and/or lower See bilirubin above. conj. degradation of hemeproteins including. Improved status of the liver. Lower heme proteindegradation is also an indication of decreased oxidative stress in thesehealthy volunteers. 5 CK, creatinine kinase On average 25% reduction invalues (N = 4). This is a clear indication of improved status of CK alsodown in higher/acute Lower also for subjects S5 and S6. skeletal musclesand heart. regime. This strong finding is due to clear increase inenergy metabolism by DGA. 6 fS-Fe On average 15% reduction in values. N= 6 Indicates increased use of Fe to the assembly of See also resultsbilirubin, aerobic heme proteins in e.g. ETS/oxidative bilirubinconjugate and phosphorylation. May be also an indication of decrease inLDH, and take into increased oxygen transporting ability to tissues byaccount head ache “hangover” erythrocytes. (Nrf2) when stopping doubleadministration (16). Absolute amount of observed decline in Ferepresents on average 0.13% of total Fe bind to blood heme proteins (2.5g). Thus observed 4 day decline could easily be explained by increasedFe binding to heme proteins due to health effects of DGA. 7 fS-GlucoseDown or unchanged (=change less than 2%) for all healthy An indicationof increased metabolic flux. Diabetes, This result is in line with latervolunteers (N = 8) in all periods insulin resistance and pancreas.results that glucose uptake is and doses; average decline only some 6%.stimulated by DGA (Example 2.3.4/glucose tolerance test). 8 fS- Tendencyfor all healthy volunteers (N = 8) is Lower risk for cardiovasculardisease. The Increased metabolic flux. The Cholesterol down but theaverage decline is very small combination of lower oxidative stress andstable or use of DGA can also in fact (only some 3-5%). reducedcholesterol in cardiovascular system may increase the intracellularreduce the risk of cardiovascular diseases efficiently. cholesterolproduction (see FIG. 4), but on the other hand plasma membrane LDL-receptors are probably also activated thus balancing the effects forfS-Cholesterol into small reduction. 9 fS-Chol.-HDL Down for all healthyvolunteers (N = 8) and in The decline is in line with the decreasedamount of Decline is in line with the all periods and doses. Averagedecline some LDL and total cholesterol. It seems to be a naturaldecreased amount of LDL and 6%. reaction on lower cholesterol. totalcholesterol. 10 fS-Chol-LDL Down or unchanged (=change less than 2%)Reduced risk of cardiovascular diseases. See also fS-Cholesterol. seecholesterol (above) for all healthy volunteers (N = 8) in all periodsand doses; average decline only some 5%. 11 fS-Lactate Down for allhealthy volunteers (N = 3) in 4 day Indicates increased viability(smaller mortality) of Improved systemic redox state dehydrogenase (LDH)test; average decline some 4%. (Subject 3 erythrocytes. Possibly also anindication of improved has been later proven in gave no meaningfulresult, see above fS-Alat systemic redox state i.e. reduced activity ofExample 2.3.3. Pyruvate for explanation.) reactions from pyruvate tolactate by lactate conversion into lactate reduces dehydrogenase (LDH).significantly with the use of DGA, which is very remarkable proof on theefficacy of DGA. 12 fS-Na Down or unchanged for all healthy volunteersLowering of blood pressure. This small decline could The decline is inline with the (Sodium) (N = 4) in 4 day test; average decline some alsobe an indication of increased metabolic flux/ increase on metabolicflux. See 1.5%. diuretic effect of DGA. It may also be an indicationalso the result that uric acid (fS- of improved renal activity. Uraat)declined unlike with many other drugs with diuretic effects. 13fS-Trigly Blood triglycerides seem to increase relatively Observed25-50% increases in blood triglycerides Increase in aerobic ATPsignificantly for most of the study subjects, for 5 subjects are likelydue to two simultaneous and production by the use of DGA althoughobserved levels are still below complementing factors, 1) due toincreased demand requires the transportation of recommended 2 mmol/lafter the increases for of triglycerides by the beta-oxidation, and 2)energy rich fatty acids from all healthy volunteers. because D-glycerategroup molecules are adipose tissues into the liver phosphorylated andthen reduced towards G-3-P. and further to be transported as Note: theincrease in endogenous triglycerides triglycerides in to e.g. skeletalconsists mostly of medium chain triglycerides that muscles, See alsoexamples 4 can be even health promoting. and 5. 14 fS-Urate Down for allhealthy volunteers (N = 6) in 4 day Decline indicates declined oxidativestress of whole This decline is in line with (uric acid) test; averagedecline some 6%. cardiovascular system. Also risk of developing goutobserved strong antioxidant decreases by declining uric acid. On theother hand properties of DGA in example 1. elevated uric acid levelshave been clearly associated Combination of increased withcardiovascular diseases, type II diabetes and metabolic flux (Examples1.1, metabolic syndrome. 2.3.3, 3 and 4) and reduction in oxidativestress is the “sweet spot” for preventing cardio- vascular diseases. 15fS-Urea Clear tendency for decline. Average decline Decline in urea ise.g. an indication of improved renal Decline in urea production is insome 7-8%. functions. It is also an indication of increased protein linewith 10% decline in plasma synthesis. The combination of increased ATPNO (Example 2.3.3). Blood urea production from beta oxidation and cleardecline in declined also in 4.5 day test nitrogen extraction from thebody, provides the with acute dosing. conversion of fat into muscles bythe use of DGA. 16 Headache All participants that stopped 2 × 6 mg/kgThis is likely an indication that either the oxygen ATP relatedexplanation is after administration at once (N = 3) experienced (ATP) ornutrition (ATP) supply to the brain cells has clearly more convincingwhen stopping some symptoms of headache after 20-48 deteriorated afterpositive effects from DGA ceased related to all other received hoursafter last DGA administration to materialize or both. results. 17Lowering of blood pressure Effect on blood pressure has been tested inLower blood pressure indicates an enhancement on This result is also inline with scientific manner only on two healthy the aerobic activity ofskeletal muscles and heart. decrease in plasma lactate that volunteers(FIG. 12). Only one person had Possibly also the Nrf2/ARE enhanced redoxstate of has been also associated with clearly elevated blood pressure:systolic 180 erythrocytes enhance oxygen transport to the lowered bloodpressure. Likely and diastolic 104 before 2 × 6 mg/kg of DGA tissues inneed (pH related increase in 2.3- enhanced aerobic ATP twice a day.After 10 day treatment blood bisphosphoglycerate). production capacityby the use pressure declined to some 160 and 90. of DGA and increasedflow of oxygen into peripheral tissues allow the blood pressure to belowered. 18 Diuretic Sodium decline in most of the study subjects.Overall, i.e. mitochondrial, cytosolic, ER and peroxisomal, metabolicflux is increased effects by the use of DGA. This likely causes diureticeffects when starting the use of DGA.

REFERENCES CITED

Eriksson C J P, Saarenmaa T, Bykoc I L, Heino P U. Acceleration ofEthanol and Acetaldehyde Oxidation by D-glycerate in Rats. Metabolism56, 895-898 (2007).

Habe H, Sato S, Fukuoka T, Kitamoto D, Sakaki K. Effect of Glyceric AcidCalcium Salt on the Viability of Ethanol-Dosed Gastric Cells. Journal ofOleo Science 60 (11), 585-590 (2011).

Hoffmann G F et al. Physiology and pathophysiology of organic acids incerebrospinal fluid. J Inherit Metab Dis. 16(4), 648-69 (1993).

Robergs R A. Exercise-Induced Metabolic Acidosis: Where do the Protonscome from? (2001) Sportscience 5(2), sportsci.org/jour/0102/rar.htm,2001.

1-22. (canceled)
 23. A method of increasing direct or indirectmitochondrial activity, RNA expression of genes encoding ETS relatedgenes, TCA activity, and/or biogenesis of new mitochondria in a subjectcomprising administering a composition comprising an effective amount ofone or more compounds selected from the group consisting of D-glycericacid, DL-glyceric acid, L-glyceric acid, and hydroxypyruvic acid, andsalts and esters thereof to a subject in need, wherein the compositionenhances mitochondrial ATP production and simultaneously reducesexcessive radical oxygen species formation from OXPHOS, and furthermorecan increase cellular capacity to adjust cytosolic NAD+/NADH-ratio intimely manner when needed.
 24. A method of enhancing physical training,performance and recovery from exercise in a subject comprisingadministering a composition comprising an effective amount of one ormore compounds selected from the group consisting of D-glyceric acid,DL-glyceric acid, L-glyceric acid, and hydroxypyruvic acid, and saltsand esters thereof to a subject in need, wherein the compositionenhances mitochondrial ATP production and simultaneously reducesexcessive radical oxygen species formation from OXPHOS, and furthermorecan increase cellular capacity to adjust cytosolic NAD+/NADH-ratio intimely manner when needed.
 25. The method according to claim 24,comprising administering the composition comprising one or morecompounds selected from the group consisting of D-glyceric acid,DL-glyceric acid, L-glyceric acid, and hydroxypyruvic acid and salts andesters thereof, and a pharmaceutically acceptable excipient.
 26. Themethod according to claim 24, comprising administering the compositionin a form of a solution, syrup, powder, ointment, capsule, tablet, or aninhalable preparation.
 27. The method according to claim 24, comprisingadministering the composition via a parenteral, oral, or topicalapplication route or by inhalation.
 28. The method according to claim24, comprising administering the composition via a beverage, a foodproduct, a functional food, a dietary supplement, or a nutritivesubstance.
 29. A method of increasing protein synthesis and sharpeningthe control of unfolded protein response or increasing the muscle yieldper gram of nutrition and simultaneously decreasing of fat content of ahuman or an animal, and/or decreasing nutrition consumption withoutlosing muscle mass of an animal, such as a mammal, poultry, and fish ina subject comprising administering a composition comprising an effectiveamount of one or more compounds selected from the group consisting ofD-glyceric acid, DL-glyceric acid, L-glyceric acid, and hydroxypyruvicacid, and salts and esters thereof to a subject in need, wherein thecomposition enhances mitochondrial ATP production and simultaneouslyreduces excessive radical oxygen species formation from OXPHOS, andfurthermore can increase cellular capacity to adjust cytosolicNAD+/NADH-ratio in timely manner when needed.
 30. The method accordingto claim 29, comprising administering the composition comprising one ormore compounds selected from the group consisting of D-glyceric acid,DL-glyceric acid, L-glyceric acid, and hydroxypyruvic acid and salts andesters thereof, and a pharmaceutically acceptable excipient.
 31. Themethod according to claim 29, comprising administering the compositionin a form of a solution, syrup, powder, ointment, capsule, tablet, or aninhalable preparation.
 32. The method according to claim 29, comprisingadministering the composition via a parenteral, oral, or topicalapplication route or by inhalation.
 33. The method according to claim29, comprising administering the composition via a beverage, a foodproduct, a functional food, a dietary supplement, or a nutritivesubstance.